2&#39;-substituted carba-nucleoside analogs for antiviral treatment

ABSTRACT

Provided are compounds of Formula I, 
     
       
         
         
             
             
         
       
     
     as well as pharmaceutical compositions containing compounds of Formula I and methods for treating Orthomyxoviridae virus infections by administering these compounds. The compounds, compositions, and methods provided are particularly useful for the treatment of Human Influenza virus infections.

This application claims the benefit of U.S. Provisional Application No.61/610,411, filed Mar. 13, 2012, the contents of which are incorporatedherein in its entirety.

FIELD OF THE INVENTION

The invention relates generally to compounds with antiviral activity,more particularly nucleosides active against Orthomyxoviridae virusinfections, as well as pharmaceutical compositions and methods using thesame.

BACKGROUND OF THE INVENTION

Influenza viruses of the Orthomyxoviridae family that belong to thegenera A and B are responsible for seasonal flu epidemics each year,which cause acute contagious respiratory infections. Children, the old,and people with chronic diseases are at high risk to develop severecomplications that lead to high morbidity and mortality rates (Memoli etal., Drug Discovery Today 2008, 13, 590-595). Among the three influenzagenera, type A viruses are the most virulent human pathogens that causethe most severe disease, can be transmitted to other species, and giverise to human influenza pandemics. The recent human influenza outbreakof the aggressive porcine A/H1N1 strain in 2009 has emphasized the needfor novel antiviral therapeutics. While yearly vaccination programs arecurrently used to protect populations from influenza infection, theseprograms must anticipate the virus strains that will be prevalent duringseasonal outbreaks to be effective and they do not address the problemof sudden, unanticipated influenza pandemics. Again, the recent humaninfluenza outbreak of the aggressive porcine A/H1N1 strain in 2009 is anexample of this problem.

Several anti-influenza therapeutics are now available and others areunder development (Hedlund et al., Viruses 2010, 2, 1766-1781). Amongthe currently available anti-influenza therapeutics are the M2 ionchannel blockers amantadine and rimantadine and the neuraminidaseinhibitors oseltamivir and zanamivir. However, resistance has developedto all of these medications. Therefore, there is a continuing need fornovel anti-influenza therapeutics.

Promising new anti-influenza agents with novel mechanisms of action arenow in development. Among these new agents is favipiravir, which targetsviral gene replication by inhibiting influenza RNA polymerase. However,it is still uncertain whether this investigational drug candidate willbecome available for therapy. Therefore, there is a continuing need todevelop additional compounds that inhibit influenza through thismechanism of action.

Certain ribosides of the nucleobases pyrrolo[1,2-f][1,2,4]triazine,imidazo[1,5-f][1,2,4]triazine, imidazo[1,2-f][1,2,4]triazine, and[1,2,4]triazolo[4,3-f][1,2,4]triazine have been disclosed inCarbohydrate Research 2001, 331(1), 77-82; Nucleosides & Nucleotides1996, 15(1-3), 793-807; Tetrahedron Letters 1994, 35(30), 5339-42;Heterocycles 1992, 34(3), 569-74; J. Chem. Soc. Perkin Trans. 1 1985, 3,621-30; J. Chem. Soc. Perkin Trans. 1 1984, 2, 229-38; WO 2000056734;Organic Letters 2001, 3(6), 839-842; J. Chem. Soc. Perkin Trans. 1 1999,20, 2929-2936; and J. Med. Chem. 1986, 29(11), 2231-5. However, thesecompounds have not been disclosed as useful for the treatment ofOrthomyxoviridae infections.

Ribosides of pyrrolo[1,2-f][1,2,4]triazinyl,imidazo[1,5-f][1,2,4]triazinyl, imidazo[1,2-f][1,2,4]triazinyl, and[1,2,4]triazolo[4,3-f][1,2,4]triazinyl nucleobases with antiviral,anti-HCV, and anti-RdRp activity have been disclosed by Babu,WO2008/089105 and WO2008/141079, Cho et al., WO2009/132123, and Francomet al. WO2010/002877. Butler et al., WO2009/132135, discloses anti-viralpyrrolo[1,2-f][1,2,4]triazinyl, imidazo[1,5-f][1,2,4]triazinyl,imidazo[1,2-f][1,2,4]triazinyl, and[1,2,4]triazolo[4,3-f][1,2,4]triazinyl nucleosides wherein the 1′position of the nucleoside sugar is substituted with a cyano or methylgroup. However, the effectiveness of these compounds for the treatmentof Orthomyxoviridae infections has not been disclosed.

SUMMARY OF THE INVENTION

Provided herein are compounds that inhibit viruses of theOrthomyxoviridae family. The invention also comprises compounds ofFormula I that inhibit viral nucleic acid polymerases, particularlyOrthomyxoviridae RNA-dependent RNA polymerase (RdRp), rather thancellular nucleic acid polymerases. Compounds of Formula I are useful fortreating Orthomyxoviridae infections in humans and other animals.

The first embodiment of the invention is directed to a compound ofFormula I:

or a pharmaceutically acceptable salt, solvate, or ester thereof;wherein:

each of R¹ and R⁷ is independently H, halogen, OR^(a), (C₁-C₈)haloalkyl,CN, N₃, (C₁-C₈)alkyl, (C₁-C₈)substituted alkyl, (C₂-C₈)alkenyl,(C₂-C₈)substituted alkenyl, (C₂-C₈)alkynyl or (C₂-C₈)substitutedalkynyl,

wherein the substituent is selected from the group consisting of —X,—R^(b), —OH, ═O, —OR^(b), —SR^(b), —S⁻, —NR^(b) ₂, —N⁺R^(b) ₃, ═NR^(b),—CX₃, —CN, —OCN, —SCN, —N═C═O, —NCS, —NO, —NO₂, ═N₂, —N₃, —NHC(═O)R^(b),—OCH))R^(b), —NHC(═O)NR^(b) ₂, —S(═O)₂—, —S(═O)₂OH, —S(═O)₂R^(b),—OS(═O)₂OR^(b), —S(═O)₂NR^(b) ₂, —S(═O)R^(b), —OP(═O)(ORN,—P(═O)(OR^(b))₂, —P(═O)(CO₂, —P(═O)(OH)₂, —P(O)(OR^(b))(O), —C(═O)R^(b),—C(═O)X, —C(S)R^(b), —C(O)OR^(b), —C(O)O⁻, —C(S)OR^(b), —C(O)SR^(b),—C(S)SR^(b), —C(O)NR^(b) ₂, —C(S)NR^(b) ₂, —C(═NR^(b))NR^(b) ₂, whereeach X is independently a halogen: F, Cl, Br, or I; and each R^(b) isindependently H, alkyl, aryl, arylalkyl, a heterocycle, or a protectinggroup or prodrug moiety;

R² is OR^(a);

R³ is halogen or N₃;

each R^(a) is independently H, aryl, arylalkyl, or (C₁-C₈)alkyl;

each of R⁴ and R⁵ is independently H, ═O, OR^(a), N(R^(a))₂, N₃, CN,S(O)_(n)R^(a), halogen, or (C₁-C₈)haloalkyl;

n is 0, 1 or 2; and

R⁶ is H, aryl, arylalkyl, or

wherein W¹ and W² are each, independently, OR^(a) or a group of theFormula Ia:

wherein:

-   -   each Y is independently a bond or O;    -   M2 is 0, 1 or 2;    -   each R^(x) is H, halogen or OH.

In a preferred embodiment, the compound of Formula I is represented byFormula II:

or a pharmaceutically acceptable salt, solvate, or ester thereof. Inother preferred embodiments, R¹ is H, R² is OH or O-benzyl and/or R³ isF or N₃ and, more preferably, R³ is F. In a certain embodiment of thepresent invention, R⁴ is NH₂ and R⁵ is H, F, Cl, Br, N₃, CN, CF₃, NH₂,SMe, or SO₂Me, and, in another embodiment, R⁵ is NH₂ and R⁴ is ═O, OH,OMe, Cl, Br, I, NH₂, NHMe, NHcPr or SMe. In still further preferredembodiments, R⁴ and R⁵ are both NH₂ or SMe, R⁵ is H, or R⁴ is ═O. Inother preferred embodiments, R⁶ is H, benzyl, or

wherein W² is OH and W¹ is a group of the Formula Ia:

wherein each Y is O; M2 is 2; and each R^(x) is H. In anotherembodiment, R⁷ is H or OH.

The second embodiment of the invention is directed to a pharmaceuticalcomposition comprising a therapeutically effective amount of a compoundof Formula I, II or III, as defined in the first embodiment of theinvention, and a pharmaceutically acceptable carrier or excipient. In acertain embodiment thereof, the pharmaceutical composition furthercomprises at least one additional therapeutic agent.

The third embodiment of the invention is directed to a method fortreating an Orthomyxoviridae infection in a mammal in need thereofcomprising administering a therapeutically effective amount of acompound of Formula I, II or III or a pharmaceutically acceptable salt,solvate or ester thereof, as defined in the first embodiment of theinvention. In some embodiments, the Orthomyxoviridae infection beingtreated is an Influenza virus A infection, an Influenza virus Binfection, or an Influenza virus C infection. In another embodiment, themethod comprises treating an Orthomyxoviridae infection in a mammal inneed thereof by administering a therapeutically effective amount of apharmaceutical composition comprising an effective amount of a FormulaI, II or III compound or a pharmaceutically acceptable salt, solvate orester thereof in combination with a pharmaceutically acceptable diluentor carrier.

In another embodiment, the present invention provides a method ofinhibiting an Orthomyxoviridae RNA-dependent RNA polymerase. In afurther embodiment, this method comprises contacting a cell infectedwith Orthomyxoviridae virus with an effective amount of a compound ofFormula I, II or III or a pharmaceutically acceptable salt, solvate, orester thereof.

In another aspect of this embodiment, the present invention provides amethod of treating an Orthomyxoviridae virus infection and, in a certainembodiment, further comprises administering a therapeutically effectiveamount of at least one additional therapeutic agent or compositionthereof selected from the group consisting of a corticosteroid, ananti-inflammatory signal transduction modulator, a β2-adrenoreceptoragonist bronchodilator, an anticholinergic, a mucolytic agent,hypertonic saline, an agent that inhibits migration of pro-inflammatorycells to the site of infection, and mixtures thereof. In certainembodiments, the additional therapeutic agent is a viral haemagglutinininhibitor, a viral neuramidase inhibitor, a M2 ion channel inhibitor, aOrthomyxoviridae RNA-dependent RNA polymerase inhibitor or a sialidase.In another embodiment, the additional therapeutic agent is selected fromthe group consisting of ribavirin, oseltamivir, zanamivir, laninamivir,peramivir, amantadine, rimantadine, CS-8958, favipiravir, AVI-7100,alpha-1 protease inhibitor and DAS 181.

In another embodiment of the invention, the compound of Formula I, II orIII and/or at least one additional therapeutic agent is administered byinhalation.

DETAILED DESCRIPTION OF THE INVENTION

The first embodiment of the present invention is directed to a compoundof Formula I:

or a pharmaceutically acceptable salt, solvate, or ester thereof;

wherein:

each of R¹ and R⁷ is independently H, halogen, Or, (C₁-C₈)haloalkyl, CN,N₃, (C₁-C₈)alkyl, (C₁-C₈)substituted alkyl, (C₂-C₈)alkenyl,(C₂-C₈)substituted alkenyl, (C₂-C₈)alkynyl or (C₂-C₈)substitutedalkynyl,

wherein the substituent is selected from the group consisting of —X,—R^(b), —OH, ═O, —OR^(b), —SR^(b), —S⁻, —NR^(b) ₂, —N⁺R^(b) ₃, ═NR^(b),—CX₃, —CN, —OCN, —SCN, —N═C═O, —NCS, —NO, —NO₂, ═N₂, —N₃, —NHC(═O)R^(b),—OC(D)R^(b), —NHC(═O)NR^(b) ₂, —S(═O)₂—, —S(═O)₂OH, —S(═O)₂R^(b),—OS(═O)₂OR^(b), —S(═O)₂NR^(b) ₂, —S(═O)R^(b), —OP(═O)(OR^(b))₂,—P(═O)(OR^(b))₂, —P(═O)(O⁻)₂, —P(═O)(OH)₂, —P(O)(OR^(b))(O),—C(═O)R^(b), —C(═O)X, —C(S)R″, —C(O)OR^(b), —C(O)O⁻, —C(S)OR^(b),—C(O)SR^(b), —C(S)SR^(b), —C(O)NR^(b) ₂, —C(S)NR^(b) ₂,—C(═NR^(b))NR^(b) ₂, where each X is independently a halogen: F, Cl, Br,or I; and each R^(b) is independently H, alkyl, aryl, arylalkyl, aheterocycle, or a protecting group or prodrug moiety;

R² is OR^(a);

R³ is halogen or N₃;

each R^(a) is independently H, aryl, arylalkyl, or (C₁-C₈)alkyl;

each of R⁴ and R⁵ is independently H, ═O, OR^(a), N(R^(a))₂, N₃, CN,S(O)_(n)R^(a), halogen, or (C₁-C₈)haloalkyl;

each n is 0, 1 or 2; and

R⁶ is H, aryl, arylalkyl, or

wherein W¹ and W² are each, independently, OR^(a) or a group of theFormula Ia:

wherein:

-   -   each Y is independently a bond or O;    -   M2 is 0, 1 or 2; and    -   each R^(x) is H, halogen or OH. In regard to Formula Ia, when Y        is O, R^(x) is not halogen.

Unless stated otherwise, the following terms and phrases as used hereinare intended to have the following meanings.

When trade names are used herein, applicants intend to independentlyinclude the tradename product and the active pharmaceuticalingredient(s) of the tradename product.

“Alkyl” is hydrocarbon containing normal, secondary, tertiary or cycliccarbon atoms. For example, an alkyl group can have 1 to 20 carbon atoms(i.e, C₁-C₂₀ alkyl), 1 to 8 carbon atoms (i.e., C₁-C₈ alkyl), or 1 to 6carbon atoms (i.e., C₁-C₆ alkyl). Examples of suitable alkyl groupsinclude, but are not limited to, methyl (Me, —CH₃), ethyl (Et, —CH₂CH₃),1-propyl (n-Pr, n-propyl, —CH₂CH₂CH₃), 2-propyl (i-Pr, i-propyl,—CH(CH₃)₂), cyclopropyl (c-propyl, cPr), 1-butyl (n-Bu, n-butyl,—CH₂CH₂CH₂CH₃), 2-methyl-1-propyl (i-Bu, i-butyl, —CH₂CH(CH₃)₂), 2-butyl(s-Bu, s-butyl, —CH(CH₃)CH₂CH₃), 2-methyl-2-propyl (t-Bu, t-butyl,—C(CH₃)₃), 1-pentyl (n-pentyl, —CH₂CH₂CH₂CH₂CH₃), 2-pentyl(—CH(CH₃)CH₂CH₂CH₃), 3-pentyl (—CH(CH₂CH₃)₂), 2-methyl-2-butyl(—C(CH₃)₂CH₂CH₃), 3-methyl-2-butyl (—CH(CH₃)CH(CH₃)₂), 3-methyl-1-butyl(—CH₂CH₂CH(CH₃)₂), 2-methyl-1-butyl (—CH₂CH(CH₃)CH₂CH₃), 1-hexyl(—CH₂CH₂CH₂CH₂CH₂CH₃), 2-hexyl (—CH(CH₃)CH₂CH₂CH₂CH₃), 3-hexyl(—CH(CH₂CH₃)(CH₂CH₂CH₃)), 2-methyl-2-pentyl (—C(CH₃)₂CH₂CH₂CH₃),3-methyl-2-pentyl (—CH(CH₃)CH(CH₃)CH₂CH₃), 4-methyl-2-pentyl(—CH(CH₃)CH₂CH(CH₃)₂), 3-methyl-3-pentyl (—C(CH₃)(CH₂CH₃)₂)_(,)2-methyl-3-pentyl (—CH(CH₂CH₃)CH(CH₃)₂), 2,3-dimethyl-2-butyl(—C(CH₃)₂CH(CH₃)₂), 3,3-dimethyl-2-butyl (—CH(CH₃)C(CH₃)₃, and octyl(—(CH₂)₇CH₃).

“Alkenyl” is a hydrocarbon containing normal, secondary, tertiary orcyclic carbon atoms with at least one site of unsaturation, i.e., acarbon-carbon, sp² double bond. For example, an alkenyl group can have 2to 20 carbon atoms (i.e., C₂-C₂₀ alkenyl), 2 to 8 carbon atoms (i.e.,C₂-C₈ alkenyl), or 2 to 6 carbon atoms (i.e., C₂-C₆ alkenyl). Examplesof suitable alkenyl groups include, but are not limited to, ethylene orvinyl (—CH═CH₂), allyl (—CH₂CH═CH₂), cyclopentenyl (—C₅H₇), and5-hexenyl (—CH₂CH₂CH₂CH₂CH═CH₂).

“Alkynyl” is a hydrocarbon containing normal, secondary, tertiary orcyclic carbon atoms with at least one site of unsaturation, i.e. acarbon-carbon, sp triple bond. For example, an alkynyl group can have 2to 20 carbon atoms (i.e., C₂-C₂₀ alkynyl), 2 to 8 carbon atoms (i.e.,C₂-C₈ alkyne,), or 2 to 6 carbon atoms (i.e., C₂-C₆ alkynyl). Examplesof suitable alkynyl groups include, but are not limited to, acetylenic(—C≡CH), propargyl (—CH₂C≡CH), and the like.

“Aryl” means an aromatic hydrocarbon radical derived by the removal ofone hydrogen atom from a single carbon atom of a parent aromatic ringsystem. For example, an aryl group can have 6 to 20 carbon atoms, 6 to14 carbon atoms, or 6 to 10 carbon atoms. Typical aryl groups include,but are not limited to, radicals derived from benzene (e.g., phenyl),substituted benzene, naphthalene, anthracene, biphenyl, and the like.

“Arylalkyl” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with an aryl radical. Typical arylalkyl groupsinclude, but are not limited to, benzyl, 2-phenylethan-1-yl,naphthylmethyl, 2-naphthylethan-1-yl, naphthobenzyl,2-naphthophenylethan-1-yl and the like. The arylalkyl group can comprise7 to 20 carbon atoms, e.g., the alkyl moiety is 1 to 6 carbon atoms andthe aryl moiety is 6 to 14 carbon atoms.

“Carbocycle” or “carbocyclyl” refers to a saturated (i.e., cycloalkyl),partially unsaturated (e.g., cycloakenyl, cycloalkadienyl, etc.) oraromatic ring having 3 to 7 carbon atoms as a monocycle, 7 to 12 carbonatoms as a bicycle, and up to about 20 carbon atoms as a polycycle.Monocyclic carbocycles have 3 to 7 ring atoms, still more typically 5 or6 ring atoms. Bicyclic carbocycles have 7 to 12 ring atoms, e.g.,arranged as a bicyclo[4,5], [5,5], [5,6] or [6,6] system, or 9 or 10ring atoms arranged as a bicyclo[5,6] or [6,6] system, or spiro-fusedrings. Non-limiting examples of monocyclic carbocycles includecyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl,1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl,1-cyclohex-2-enyl, 1-cyclohex-3-enyl, and phenyl. Non-limiting examplesof bicyclo carbocycles includes naphthyl, tetrahydronapthalene, anddecaline.

“Haloalkyl” is an alkyl group, as defined above, in which one or morehydrogen atoms of the alkyl group is replaced with a halogen atom. Thealkyl portion of a haloalkyl group can have 1 to 20 carbon atoms (i.e.,C₁-C₂₀ haloalkyl), 1 to 12 carbon atoms (i.e., C₁-C₁₂ haloalkyl), or 1to 6 carbon atoms (i.e., C₁-C₆ alkyl). Examples of suitable haloalkylgroups include, but are not limited to, —CF₃, —CHF₂, —CFH₂, —CH₂CF₃, andthe like.

“Heterocycle” or “heterocyclyl” as used herein includes by way ofexample and not limitation those heterocycles described in Paquette, LeoA.; Principles of Modern Heterocyclic Chemistry (W. A. Benjamin, NewYork, 1968), particularly Chapters 1, 3, 4, 6, 7, and 9; The Chemistryof Heterocyclic Compounds, A Series of Monographs” (John Wiley & Sons,New York, 1950 to present), in particular Volumes 13, 14, 16, 19, and28; and J. Am. Chem. Soc. (1960) 82:5566. In one specific embodiment ofthe invention “heterocycle” includes a “carbocycle” as defined herein,wherein one or more (e.g., 1, 2, 3, or 4) carbon atoms have beenreplaced with a heteroatom (e.g., O, N, or S). The terms “heterocycle”or “heterocyclyl” includes saturated rings, partially unsaturated rings,and aromatic rings (i.e., heteroaromatic rings).

The term “substituted” in reference to alkyl, alkenyl, aryl, etc., forexample, “substituted alkyl”, “substituted alkenyl”, “substituted aryl”,respectively, in which one or more hydrogen atoms are each independentlyreplaced with a non-hydrogen substituent. Typical substituents include,but are not limited to, —X, —R^(b), —OH, ═O, —OR^(b), —SR^(b), —S⁻,—NR^(b) ₂, —N⁺R^(b) ₃, ═NR^(b), —CX₃, —CN, —OCN, —SCN, —N═C═O, —NCS,—NO, —NO₂, ═N₂, —N₃, —NHC(═O)R^(b), —OC(═O)R^(b), —NHC(═O)NR^(b) ₂,—S(═O)₂—, —S(═O)₂OH, —S(═O)₂R^(b), —OS(═O)₂OR^(b), —S(═O)₂NR^(b) ₂,—S(═O)R^(b), —OP(═O)(OR^(b))₂, —P(═O)(OR^(b))₂, —P(═O)(O⁻)₂,—P(═O)(OH)₂, —P(O)(OR^(b))(O), —C(═O)R^(b), —C(═O)X, —C(S)R^(b),—C(O)OR^(b), —C(O)O⁻, —C(S)OR^(b), —C(O)SR^(b), —C(S)SR^(b), —C(O)NR^(b)₂, —C(S)NR^(b) ₂, —C(═NR^(b))NR^(b) ₂, where each X is independently ahalogen: F, Cl, Br, or I; and each R^(b) is independently H, alkyl,aryl, arylalkyl, a heterocycle, or a protecting group or prodrug moiety.

The term “prodrug” as used herein refers to any compound that whenadministered to a biological system generates the drug substance, i.e.,active ingredient, as a result of spontaneous chemical reaction(s),enzyme catalyzed chemical reaction(s), photolysis, and/or metabolicchemical reaction(s). A prodrug is thus a covalently modified analog orlatent form of a therapeutically active compound.

“Protecting group” refers to a moiety of a compound that masks or altersthe properties of a functional group or the properties of the compoundas a whole. The chemical substructure of a protecting group varieswidely. One function of a protecting group is to serve as anintermediate in the synthesis of the parental drug substance. Chemicalprotecting groups and strategies for protection/deprotection are wellknown in the art. See: “Protective Groups in Organic Chemistry”,Theodora W. Greene (John Wiley & Sons, Inc., New York, 1991. Protectinggroups are often utilized to mask the reactivity of certain functionalgroups, to assist in the efficiency of desired chemical reactions, e.g.,making and breaking chemical bonds in an ordered and planned fashion.Protection of functional groups of a compound alters other physicalproperties besides the reactivity of the protected functional group,such as the polarity, lipophilicity (hydrophobicity), and otherproperties which can be measured by common analytical tools. Chemicallyprotected intermediates may themselves be biologically active orinactive.

Protected compounds may also exhibit altered, and in some cases,optimized properties in vitro and in vivo, such as passage throughcellular membranes and resistance to enzymatic degradation orsequestration. In this role, protected compounds with intendedtherapeutic effects may be referred to as prodrugs. Another function ofa protecting group is to convert the parental drug into a prodrug,whereby the parental drug is released upon conversion of the prodrug invivo. Because active prodrugs may be absorbed more effectively than theparental drug, prodrugs may possess greater potency in vivo than theparental drug. Protecting groups are removed either in vitro, in theinstance of chemical intermediates, or in vivo, in the case of prodrugs.With chemical intermediates, it is not particularly important that theresulting products after deprotection, e.g., alcohols, bephysiologically acceptable, although in general it is more desirable ifthe products are pharmacologically innocuous.

“Prodrug moiety” means a labile functional group which separates fromthe active inhibitory compound during metabolism, systemically, inside acell, by hydrolysis, enzymatic cleavage, or by some other process(Bundgaard, Hans, “Design and Application of Prodrugs” in Textbook ofDrug Design and Development (1991), P. Krogsgaard-Larsen and H.Bundgaard, Eds. Harwood Academic Publishers, pp. 113-191). Enzymes whichare capable of an enzymatic activation mechanism with the phosphonateprodrug compounds of the invention include, but are not limited to,amidases, esterases, microbial enzymes, phospholipases, cholinesterases,and phosphases. Prodrug moieties can serve to enhance solubility,absorption and lipophilicity to optimize drug delivery, bioavailabilityand efficacy. A prodrug moiety may include an active metabolite or drugitself.

Exemplary prodrug moieties include the hydrolytically sensitive orlabile acyloxymethyl esters —CH₂OC(═O)R³⁰ and acyloxymethyl carbonates—CH₂C(═O)OR³⁰ where R³⁰ is C₁-C₆ alkyl, C₁-C₆ substituted alkyl, C₆-C₂₀aryl or C₆-C₂₀ substituted aryl. The acyloxyalkyl ester was used as aprodrug strategy for carboxylic acids and then applied to phosphates andphosphonates by Farquhar et al., (1983) J. Pharm. Sci. 72: 324; alsoU.S. Pat. Nos. 4,816,570, 4,968,788, 5663159 and 5792756. In certaincompounds of the invention, a prodrug moiety is part of a phosphategroup. The acyloxyalkyl ester may be used to deliver phosphoric acidsacross cell membranes and to enhance oral bioavailability. A closevariant of the acyloxyalkyl ester, the alkoxycarbonyloxyalkyl ester(carbonate), may also enhance oral bioavailability as a prodrug moietyin the compounds of the combinations of the invention. An exemplaryacyloxymethyl ester is pivaloyloxymethoxy, (POM)—CH₂C(═O)C(CH₃)₃. Anexemplary acyloxymethyl carbonate prodrug moiety ispivaloyloxymethylcarbonate (POC)—CH₂C(═O)OC(CH₃)₃.

The phosphate group may be a phosphate prodrug moiety. The prodrugmoiety may be sensitive to hydrolysis, such as, but not limited to thosecomprising a pivaloyloxymethyl carbonate (POC) or POM group.Alternatively, the prodrug moiety may be sensitive to enzymaticpotentiated cleavage, such as a lactate ester or a phosphonamidate-estergroup.

One skilled in the art will recognize that substituents and othermoieties of the compounds of Formula I should be selected in order toprovide a compound which is sufficiently stable to provide apharmaceutically useful compound which can be formulated into anacceptably stable pharmaceutical composition. Compounds of Formula Iwhich have such stability are contemplated as falling within the scopeof the present invention.

It is to be noted that all enantiomers, diastereomers, and racemicmixtures, tautomers, polymorphs, pseudopolymorphs of compounds withinthe scope of Formula I and pharmaceutically acceptable salts (as well ascomplexes, co-crystals, etc.), solvates or esters thereof are embracedby the present invention. All mixtures of such enantiomers anddiastereomers are within the scope of the present invention.

A compound of Formula I and its pharmaceutically acceptable salts,solvates, or esters may exist as different polymorphs orpseudopolymorphs. As used herein, crystalline polymorphism means theability of a crystalline compound to exist in different crystalstructures. The crystalline polymorphism may result from differences incrystal packing (packing polymorphism) or differences in packing betweendifferent conformers of the same molecule (conformational polymorphism).As used herein, crystalline pseudopolymorphism means the ability of ahydrate or solvate of a compound to exist in different crystalstructures. The pseudopolymorphs of the instant invention may exist dueto differences in crystal packing (packing pseudopolymorphism) or due todifferences in packing between different conformers of the same molecule(conformational pseudopolymorphism). The instant invention comprises allpolymorphs and pseudopolymorphs of the compounds of Formula I and theirpharmaceutically acceptable salts.

A compound of Formula I and its pharmaceutically acceptable salts,solvates or esters may also exist as an amorphous solid. As used herein,an amorphous solid is a solid in which there is no long-range order ofthe positions of the atoms in the solid. This definition applies as wellwhen the crystal size is two nanometers or less. Additives, includingsolvents, may be used to create the amorphous forms of the instantinvention. The instant invention comprises all amorphous forms of thecompounds of Formula I and their pharmaceutically acceptable salts.

In a certain embodiment of the invention, the compound of Formula I isrepresented by Formula II:

or a pharmaceutically acceptable salt, solvate, or ester thereof;

wherein the variables are as defined for Formula I. Preferably, R¹ inFormula II is H, R⁴ is NH₂ or ═O, and/or R⁵ is NH₂ or H. Morepreferably, the compound is selected from the group consisting of

or a pharmaceutically acceptable salt, solvate, or ester thereof.

In a preferred embodiment of the compounds of Formula I, R¹ is H, CH₂OH,CH₂F, CHF₂, CH═CH₂, C≡CH, CN, CH₂CH═CH₂, N₃, CH₃ or CH₂CH₃, and, morepreferably, R¹ is H.

In a further embodiment of the invention, R² is OH or O-benzyl, and,more preferably is OH.

In a further embodiment of the invention, R³ is F or N₃, and, morepreferably R³ is F.

In a further preferred embodiment, R⁴ and R⁵ are selected from H, NH₂,═O, NHMe, NHcPr, OH, OMe, Cl, Br, I, SMe, F, N₃, CN, CF₃, and SO₂Me, andmore preferably R⁴ is ═O or NH₂, and/or R⁵ is H or NH₂.

In a further embodiment, R⁶ is H, benzyl, or

wherein W² is OH and W¹ is a group of the Formula Ia:

wherein:

Y is O;

M2 is 2; and

each R^(x) is H, and, more preferably, R⁶ is H.

In a further embodiment of the invention, R⁷ is H or OH, and, morepreferably, R⁷ is H.

In other preferred embodiments of the invention, R¹ is H, R² is OH andR³ is F. In another preferred embodiment, R¹ is H, R² is OH, R³ is F, R⁴and R⁵ are NH₂, H or ═O, and R⁶ and R⁷ are hydrogen.

In still another preferred embodiment, R¹ is H, R² is O-benzyl or OH, R³is F, R⁴ is SMe, NH₂ or ═O, R⁵ is SMe, SO₂Me, H or NH₂, R⁶ is benzyl or

wherein W² is OH and W¹ is a group of the Formula Ia:

wherein:

Y is O; M2 is 2; and

each R^(x) is H, and R⁷ is H or OH.

In other certain embodiments of the invention, R⁴ is NH₂ and R⁵ is H, F,Cl, Br, N₃, CN, CF₃, NH₂, SMe, or SO₂Me, or R⁵ is NH₂ and R⁴ is ═O, OH,OMe, Cl, Br, I, NH₂, NHMe, NHcPr or SMe. In preferred embodimentsthereof, R⁴ and R⁵ are both NH₂ or SMe, R⁵ is H, or R⁴ is ═O.

In another embodiment of the invention, R¹ is H, R² is O-benzyl, R³ isF, R⁴ is SMe, NH₂, OMe or OCH₂CH₃, R⁵ is H, SMe, SO₂Me, NH₂, N₃ or F, R⁶is benzyl, and R⁷ is H or OH.

In a preferred embodiment of the invention, the compound of Formula Iis:

or a pharmaceutically acceptable salt, solvate, or ester thereof.

In another embodiment of the invention, the compound of Formula I is:

or a pharmaceutically acceptable salt, solvate, or ester thereof.

In a still further preferred embodiment of the invention, the compoundof Formula I is:

or a pharmaceutically acceptable salt, solvate, or ester thereof.

In a certain embodiment of the invention, the present invention isdirected to compounds of Formula III:

wherein

R⁸ is NH₂, OMe, OCH₂CH₃ or ═O and R⁹ is NH₂, H, or F,

or a pharmaceutically acceptable salt, solvate, or ester thereof.

Preferably, the compound of Formula III is selected from the group

consisting of

or a pharmaceutically acceptable salt, solvate, or ester thereof. Morepreferably, the compound of Formula III is selected from the groupconsisting of

or a pharmaceutically acceptable salt, solvate, or ester thereof.

The second embodiment of the invention is directed to a pharmaceuticalcomposition comprising a therapeutically effective amount of a compoundof Formula I, as defined above with respect to the first embodiment ofthe invention, and a pharmaceutically acceptable carrier or excipient.In a certain embodiment thereof, the compound of Formula I isrepresented by Formula II or Formula III, as defined above with respectto the first embodiment of the invention. Terms in the second embodimentof the invention are defined as above with respect to the firstembodiment of the invention. Preferred embodiments of R¹, R², R³, R⁴,R⁵, R⁶ and R⁷ in the second embodiment of the invention are the same asfor the first embodiment of the invention.

In a preferred embodiment of the second embodiment of the invention, thecompound of Formula I is:

or a pharmaceutically acceptable salt, solvate, or ester thereof, andmore preferably is

or a pharmaceutically acceptable salt, solvate, or ester thereof.

In another preferred embodiment of the invention, the compound ofFormula I is:

or a pharmaceutically acceptable salt, solvate, or ester thereof.

The terms “pharmaceutical composition” and “pharmaceutical formulation”are used interchangeably herein. Pharmaceutical compositions of thepresent invention contain compounds of this invention and may beformulated using any conventional carriers and excipients, which will beselected in accord with ordinary practice. Tablets will containexcipients, glidants, fillers, binders and the like. Aqueouspharmaceutical formulations are prepared in sterile form, and, whenintended for delivery by other than oral administration, generally willbe isotonic. All pharmaceutical formulations will optionally containexcipients such as those set forth in the “Handbook of PharmaceuticalExcipients” (1986). Suitable excipients include, but are not limited to,ascorbic acid and other antioxidants, chelating agents, such as EDTA,carbohydrates, such as dextran, hydroxyalkylcellulose,hydroxyalkylmethylcellulose, stearic acid and the like. The pH of thepharmaceutical formulations may preferably range from about 3 to about11, and more preferably from about 7 to about 10.

While it is possible for the active ingredients to be administeredalone, it may be preferable to present them as pharmaceuticalformulations. The pharmaceutical formulations, both for veterinary andfor human use, of the invention comprise at least one active ingredient,as above defined, together with one or more carriers or excipients andoptionally additional therapeutic agents.

A therapeutically effective amount or effective dose are usedinterchangeably herein and are understood to mean the amount of activeingredient required to bring about the desired result. The effectivedose of active ingredient depends, at least, on the nature of thecondition being treated, toxicity, whether the compound is being usedprophylactically (lower doses) or against an active viral infection, themethod of delivery, and the pharmaceutical formulation, and can readilybe determined by the clinician using conventional dose escalationstudies. The effective amount may be about 0.0001 to about 100 mg/kgbody weight per day; preferably, from about 0.01 to about 10 mg/kg bodyweight per day; more preferably, from about 0.01 to about 5 mg/kg bodyweight per day; and most preferably, from about 0.05 to about 0.5 mg/kgbody weight per day. For example, the daily candidate dose for an adulthuman of approximately 70 kg body weight may range from about 1 mg toabout 1000 mg, preferably between about 5 mg and about 500 mg, and maytake the form of single or multiple doses.

In another embodiment, the pharmaceutical composition further comprisesat least one additional therapeutic agent. The additional therapeuticagent may be another compound of Formula I or any therapeutic agentsuitable for use with the Formula I compound. For example, theadditional therapeutic agent may be selected from the group consistingof a corticosteroid, an anti-inflammatory signal transduction modulator,a β2-adrenoreceptor agonist bronchodilator, an anticholinergic, amucolytic agent, hypertonic saline, an agent that inhibits migration ofpro-inflammatory cells to the site of infection, and mixtures thereof.The additional therapeutic agent may also include other drugs fortreating Orthomyxoviridae virus infections. In other embodiments, theadditional therapeutic agent may be viral haemagglutinin inhibitors,viral neuramidase inhibitors, M2 ion channel blockers, OrthomyxoviridaeRNA-dependent RNA polymerases inhibitors, sialidases, and other drugsfor treating Orthomyxoviridae infections. In still yet anotherembodiment, the additional therapeutic agent is an interferon,ribavirin, oseltamivir, zanamivir, laninamivir, peramivir, amantadine,rimantadine, CS-8958, favipiravir, AVI-7100, alpha-1 protease inhibitoror DAS 181.

In other certain embodiments of the invention, the Orthomyxoviridaeinfection being treated by the pharmaceutical composition is caused by aInfluenza A virus, a Influenza B virus or a Influenza C virus.

The pharmaceutical compositions include those suitable for anyadministration route appropriate to the condition to be treated.Suitable routes include oral, inhalation, rectal, nasal, topical(including buccal and sublingual), vaginal and parenteral (includingsubcutaneous, intramuscular, intravenous, intradermal, intrathecal andepidural), and the like. It will be appreciated that the preferred routemay vary, for example, with the condition of the recipient.

The pharmaceutical compositions may conveniently be presented in unitdosage form and may be prepared by any method known in the art ofpharmacy. Techniques and pharmaceutical compositions generally are foundin Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton,Pa.). Such methods include the step of bringing into association theactive ingredient with the carrier which constitutes one or moreaccessory ingredients. In general, the pharmaceutical compositions areprepared by uniformly and intimately bringing into association theactive ingredient with liquid carriers or finely divided solid carriersor both, and then, if necessary, shaping the product.

Pharmaceutical compositions of the present invention suitable for oraladministration may be presented as discrete units such as capsules,cachets or tablets each containing a predetermined amount of the activeingredient; as a powder or granules; as a solution or a suspension in anaqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion ora water-in-oil liquid emulsion. The active ingredient may also beadministered as a bolus, electuary or paste.

A tablet is made by compression or molding, optionally with one or moreaccessory ingredients. Compressed tablets may be prepared by compressingin a suitable machine the active ingredient in a free-flowing form suchas a powder or granules, optionally mixed with a binder, lubricant,inert diluent, preservative, surface active or dispersing agent. Moldedtablets may be made by molding in a suitable machine a mixture of thepowdered active ingredient moistened with an inert liquid diluent. Thetablets may optionally be coated or scored and optionally are formulatedso as to provide slow or controlled release of the active ingredienttherefrom.

For infections of the eye or other external tissues e.g. mouth and skin,the pharmaceutical compositions are preferably applied as a topicalointment or cream containing the active ingredient(s) in an amount of,for example, 0.075 to 20% w/w (including active ingredient(s) in a rangebetween 0.1% and 20% in increments of 0.1% w/w such as 0.6% w/w, 0.7%w/w, etc.), preferably 0.2 to 15% w/w and most preferably 0.5 to 10%w/w. When formulated in an ointment, the active ingredients may beemployed with either a paraffinic or a water-miscible ointment base.Alternatively, the active ingredients may be formulated in a cream withan oil-in-water cream base.

If desired, the aqueous phase of the cream base may include, forexample, at least 30% w/w of a polyhydric alcohol, i.e. an alcoholhaving two or more hydroxyl groups such as propylene glycol, butane1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol(including PEG 400) and mixtures thereof. The topical pharmaceuticalcompositions may desirably include a compound which enhances absorptionor penetration of the active ingredient through the skin or otheraffected areas. Examples of such dermal penetration enhancers includedimethyl sulphoxide and related analogs.

The oily phase of the emulsions of this invention may be constitutedfrom known ingredients in a known manner. While the phase may comprisemerely an emulsifier (otherwise known as an emulgent), it desirablycomprises a mixture of at least one emulsifier with a fat or an oil orwith both a fat and an oil. Preferably, a hydrophilic emulsifier isincluded together with a lipophilic emulsifier, which acts as astabilizer. It is also preferred to include both an oil and a fat.Together, the emulsifier(s) with or without stabilizer(s) make up theso-called emulsifying wax, and the wax together with the oil and fatmake up the so-called emulsifying ointment base which forms the oilydispersed phase of the cream pharmaceutical compositions.

Emulgents and emulsion stabilizers suitable for use in thepharmaceutical composition of the invention include, but are not limitedto, Tween® 60, Span® 80, cetostearyl alcohol, benzyl alcohol, myristylalcohol, glyceryl mono-stearate and sodium lauryl sulfate.

The choice of suitable oils or fats for the pharmaceutical compositionis based on achieving the desired cosmetic properties. The cream shouldpreferably be a non-greasy, non-staining and washable product withsuitable consistency to avoid leakage from tubes or other containers.Straight or branched chain, mono- or dibasic alkyl esters such asdi-isoadipate, isocetyl stearate, propylene glycol diester of coconutfatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate,butyl stearate, 2-ethylhexyl palmitate or a blend of branched chainesters known as Crodamol CAP may be used, the last three being preferredesters. These may be used alone or in combination depending on theproperties required. Alternatively, high melting point lipids such aswhite soft paraffin and/or liquid paraffin or other mineral oils areused.

Pharmaceutical compositions according to the present invention comprisea combination according to the invention together with one or morepharmaceutically acceptable carriers or excipients and optionallyadditional therapeutic agents. Pharmaceutical compositions containingthe active ingredient may be in any form suitable for the intendedmethod of administration. When used for oral use for example, tablets,troches, lozenges, aqueous or oil suspensions, dispersible powders orgranules, emulsions, hard or soft capsules, syrups or elixirs may beprepared. Pharmaceutical compositions intended for oral use may beprepared according to any method known to the art for the manufacture ofpharmaceutical compositions and such compositions may contain one ormore agents including sweetening agents, flavoring agents, coloringagents and preserving agents, in order to provide a palatablepreparation. Tablets containing the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipient which are suitable formanufacture of tablets are acceptable. These excipients may be, forexample, inert diluents, such as calcium or sodium carbonate, lactose,calcium or sodium phosphate; granulating and disintegrating agents, suchas maize starch, or alginic acid; binding agents, such as starch,gelatin or acacia; and lubricating agents, such as magnesium stearate,stearic acid or talc. Tablets may be uncoated or may be coated by knowntechniques including microencapsulation to delay disintegration andadsorption in the gastrointestinal tract and thereby provide a sustainedaction over a longer period. For example, a time delay material such asglyceryl monostearate or glyceryl distearate alone or with a wax may beemployed.

Pharmaceutical compositions for oral use may be also presented as hardgelatin capsules where the active ingredient is mixed with an inertsolid diluent, for example calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, such as peanut oil, liquid paraffin or olive oil.

Aqueous suspensions of the invention contain the active materials inadmixture with excipients suitable for the manufacture of aqueoussuspensions. Such excipients include a suspending agent, such as sodiumcarboxymethylcellulose, methylcellulose, hydroxypropyl methylcelluose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia,and dispersing or wetting agents such as a naturally-occurringphosphatide (e.g., lecithin), a condensation product of an alkyleneoxide with a fatty acid (e.g., polyoxyethylene stearate), a condensationproduct of ethylene oxide with a long chain aliphatic alcohol (e.g.,heptadecaethyleneoxycetanol), a condensation product of ethylene oxidewith a partial ester derived from a fatty acid and a hexitol anhydride(e.g., polyoxyethylene sorbitan monooleate). The aqueous suspension mayalso contain one or more preservatives such as ethyl or n-propylp-hydroxy-benzoate, one or more coloring agents, one or more flavoringagents and one or more sweetening agents, such as sucrose or saccharin.

Oil suspensions may be formulated by suspending the active ingredient ina vegetable oil, such as arachis oil, olive oil, sesame oil or coconutoil, or in a mineral oil such as liquid paraffin. The oral suspensionsmay contain a thickening agent, such as beeswax, hard paraffin or cetylalcohol. Sweetening agents, such as those set forth above, and flavoringagents may be added to provide a palatable oral preparation. Thesecompositions may be preserved by the addition of an antioxidant such asascorbic acid.

Dispersible powders and granules of the invention suitable forpreparation of an aqueous suspension by the addition of water providethe active ingredient in admixture with a dispersing or wetting agent, asuspending agent, and one or more preservatives. Suitable dispersing orwetting agents and suspending agents are exemplified by those disclosedabove. Additional excipients, for example sweetening, flavoring andcoloring agents, may also be present.

The pharmaceutical compositions of the invention may also be in the formof oil-in-water emulsions. The oily phase may be a vegetable oil, suchas olive oil or arachis oil, a mineral oil, such as liquid paraffin, ora mixture of these. Suitable emulsifying agents includenaturally-occurring gums, such as gum acacia and gum tragacanth,naturally-occurring phosphatides, such as soybean lecithin, esters orpartial esters derived from fatty acids and hexitol anhydrides, such assorbitan monooleate, and condensation products of these partial esterswith ethylene oxide, such as polyoxyethylene sorbitan monooleate. Theemulsion may also contain sweetening and flavoring agents. Syrups andelixirs may be formulated with sweetening agents, such as glycerol,sorbitol or sucrose. Such pharmaceutical compositions may also contain ademulcent, a preservative, a flavoring or a coloring agent.

The pharmaceutical compositions of the invention may be in the form of asterile injectable preparation, such as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according tothe known art using those suitable dispersing or wetting agents andsuspending agents which have been mentioned above. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent,such as a solution in 1,3-butane-diol or prepared as a lyophilizedpowder. Among the acceptable vehicles and solvents that may be employedare water, Ringer's solution and isotonic sodium chloride solution. Inaddition, sterile fixed oils may conventionally be employed as a solventor suspending medium. For this purpose any bland fixed oil may beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid may likewise be used in the preparation ofinjectables.

The amount of active ingredient that may be combined with a carrier, forexample, to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. For example, atime-release pharmaceutical composition intended for oral administrationto humans may contain about 1 to about 1000 mg of active ingredientcompounded with an appropriate and convenient amount of carrier whichmay vary from about 5 to about 95% of the total compositions(weight:weight). The pharmaceutical composition can be prepared toprovide easily measurable amounts for administration. For example, anaqueous solution intended for intravenous infusion may contain fromabout 3 to about 500 μg of the active ingredient per milliliter ofsolution in order to achieve an infusion rate of about 30 mL/hr.

Pharmaceutical compositions suitable for topical administration to theeye also include eye drops wherein the active ingredient is dissolved orsuspended in a suitable carrier, especially an aqueous solvent for theactive ingredient. The active ingredient is preferably present in suchpharmaceutical compositions in a concentration of about 0.5 to about 20%w/w.

Pharmaceutical compositions suitable for topical administration in themouth include lozenges comprising the active ingredient in a flavoredbasis, usually sucrose and acacia or tragacanth; pastilles comprisingthe active ingredient in an inert basis such as gelatin and glycerin, orsucrose and acacia; and mouthwashes comprising the active ingredient ina suitable liquid carrier.

Pharmaceutical compositions for rectal administration may be presentedas a suppository with a suitable base comprising for example cocoabutter or a salicylate.

Pharmaceutical compositions suitable for intrapulmonary or nasaladministration have a particle size for example in the range of about0.1 to about 500 microns, such as about 0.5, about 1, about 30, about 35etc., which is administered by rapid inhalation through the nasalpassage or by inhalation through the mouth so as to reach the alveolarsacs. Suitable pharmaceutical compositions include aqueous or oilysolutions of the active ingredient. Pharmaceutical compositions suitablefor aerosol or dry powder administration may be prepared according toconventional methods and may be delivered with additional therapeuticagents such as compounds heretofore used in the treatment or prophylaxisof Orthomyxoviridae infections as described below.

In another aspect, the invention is a novel, efficacious, safe,nonirritating and physiologically compatible inhalable compositioncomprising a compound of Formula I, II, or a pharmaceutically acceptablesalt thereof, suitable for treating Orthomyxoviridae infections andpotentially associated bronchiolitis. Preferred pharmaceuticallyacceptable salts are inorganic acid salts including hydrochloride,hydrobromide, sulfate or phosphate salts as they may cause lesspulmonary irritation. Preferably, the inhalable pharmaceuticalcomposition is delivered to the endobronchial space in an aerosolcomprising particles with a mass median aerodynamic diameter (MMAD)between about 1 and about 5 μm. Preferably, the compound of Formula I isformulated for aerosol delivery using a nebulizer, pressurized metereddose inhaler (pMDI), or dry powder inhaler (DPI).

Non-limiting examples of nebulizers include atomizing, jet, ultrasonic,pressurized, vibrating porous plate, or equivalent nebulizers includingthose nebulizers utilizing adaptive aerosol delivery technology (Denyer,J. Aerosol Medicine Pulmonary Drug Delivery 2010, 23 Supp 1, S1-S10). Ajet nebulizer utilizes air pressure to break a liquid solution intoaerosol droplets. An ultrasonic nebulizer works by a piezoelectriccrystal that shears a liquid into small aerosol droplets. A pressurizednebulization system forces solution under pressure through small poresto generate aerosol droplets. A vibrating porous plate device utilizesrapid vibration to shear a stream of liquid into appropriate dropletsizes.

In a preferred embodiment, the pharmaceutical composition fornebulization is delivered to the endobronchial space in an aerosolcomprising particles with a MMAD predominantly between about 1 μm andabout 5 μm using a nebulizer able to aerosolize the pharmaceuticalcomposition of the compound of Formula I into particles of the requiredMMAD. To be optimally therapeutically effective and to avoid upperrespiratory and systemic side effects, the majority of aerosolizedparticles should not have a MMAD greater than about 5 μm. If an aerosolcontains a large number of particles with a MMAD larger than 5 μm, theparticles are deposited in the upper airways decreasing the amount ofdrug delivered to the site of inflammation and bronchoconstriction inthe lower respiratory tract. If the MMAD of the aerosol is smaller thanabout 1 μm, then the particles have a tendency to remain suspended inthe inhaled air and are subsequently exhaled during expiration.

When formulated and delivered according to the method of the invention,the aerosol pharmaceutical composition for nebulization delivers atherapeutically efficacious dose of the compound of Formula Ito the siteof Orthomyxoviridae infection sufficient to treat the Orthomyxoviridaeinfection. The amount of drug administered must be adjusted to reflectthe efficiency of the delivery of a therapeutically effictive dose ofthe compound of Formula I. In a preferred embodiment, a combination ofthe aqueous aerosol pharmaceutical composition with the atomizing, jet,pressurized, vibrating porous plate, or ultrasonic nebulizer permits,depending on the nebulizer, about, at least, 20, to about 90%, typicallyabout 70% delivery of the administered dose of the compound of FormulaI, II or III into the airways. In a preferred embodiment, at least about30 to about 50% of the active ingredient is delivered. More preferably,about 70 to about 90% of the active ingredient is delivered.

In another embodiment of the instant invention, a compound of Formula Ior a pharmaceutically acceptable salt thereof, is delivered as a dryinhalable powder. The compounds of the invention are administeredendobronchially as a dry powder pharmaceutical composition toefficacious deliver fine particles of compound into the endobronchialspace using dry powder or metered dose inhalers. For delivery by DPI,the compound of Formula I is processed into particles with,predominantly, MMAD between about 1 μm and about 5 μm by milling spraydrying, critical fluid processing, or precipitation from solution. Mediamilling, jet milling and spray-drying devices and procedures capable ofproducing the particle sizes with a MMAD between about 1 μm and about 5μm are well know in the art. In one embodiment, excipients are added tothe compound of Formula I before processing into particles of therequired sizes. In another embodiment, excipients are blended with theparticles of the required size to aid in dispersion of the drugparticles, for example by using lactose as an excipient.

Particle size determinations are made using devices well known in theart. For example, a multi-stage Anderson cascade impactor or othersuitable method such as those specifically cited within the USPharmacopoeia Chapter 601 as characterizing devices for aerosols withinmetered-dose and dry powder inhalers.

In another preferred embodiment, a compound of Formula I is delivered asa dry powder using a device such as a dry powder inhaler or other drypowder dispersion devices. Non-limiting examples of dry powder inhalersand devices include those disclosed in U.S. Pat. No. 5,458,135; U.S.Pat. No. 5,740,794; U.S. Pat. No. 5,775,320; U.S. Pat. No. 5,785,049;U.S. Pat. No. 3,906,950; U.S. Pat. No. 4,013,075; U.S. Pat. No.4,069,819; U.S. Pat. No. 4,995,385; U.S. Pat. No. 5,522,385; U.S. Pat.No. 4,668,218; U.S. Pat. No. 4,667,668; U.S. Pat. No. 4,805,811 and U.S.Pat. No. 5,388,572. There are two major designs of dry powder inhalers.One design is a metering device in which a reservoir for the drug isplace within the device and the patient adds a dose of the drug into theinhalation chamber. The second design is a factory-metered device inwhich each individual dose has been manufactured in a separatecontainer. Both systems depend on the pharmaceutical composition of thedrug into small particles of MMAD from 1 μm and about 5 μm, and ofteninvolve co-formulation with larger excipient particles such as, but notlimited to, lactose. Drug powder is placed in the inhalation chamber(either by device metering or by breakage of a factory-metered dosage)and the inspiratory flow of the patient accelerates the powder out ofthe device and into the oral cavity. Non-laminar flow characteristics ofthe powder path cause the excipient-drug aggregates to decompose, andthe mass of the large excipient particles causes their impaction at theback of the throat, while the smaller drug particles are deposited deepin the lungs. In preferred embodiments, a compound of Formula I, or apharmaceutically acceptable salt thereof, is delivered as a dry powderusing either type of dry powder inhaler as described herein, wherein theMMAD of the dry powder, exclusive of any excipients, is predominantly inthe range of about 1 μm to about 5 μm.

In another preferred embodiment, a compound of Formula I is delivered asa dry powder using a metered dose inhaler. Non-limiting examples ofmetered dose inhalers and devices include those disclosed in U.S. Pat.No. 5,261,538; U.S. Pat. No. 5,544,647; U.S. Pat. No. 5,622,163; U.S.Pat. No. 4,955,371; U.S. Pat. No. 3,565,070; U.S. Pat. No. 3,361,306 andU.S. Pat. No. 6,116,234. In preferred embodiments, a compound of FormulaI, or a pharmaceutically acceptable salt thereof, is delivered as a drypowder using a metered dose inhaler wherein the MMAD of the dry powder,exclusive of any excipients, is predominantly in the range of about 1 toabout 5 μm.

Pharmaceutical compositions suitable for vaginal administration may bepresented as pessaries, tampons, creams, gels, pastes, foams or spraypharmaceutical compositions containing in addition to the activeingredient such carriers as are known in the art to be appropriate.

Pharmaceutical compositions suitable for parenteral administrationinclude aqueous and non-aqueous sterile injection solutions which maycontain anti-oxidants, buffers, bacteriostats and solutes which renderthe pharmaceutical composition isotonic with the blood of the intendedrecipient; and aqueous and non-aqueous sterile suspensions which mayinclude suspending agents and thickening agents.

The pharmaceutical compositions are presented in unit-dose or multi-dosecontainers, for example sealed ampoules and vials, and may be stored ina freeze-dried (lyophilized) condition requiring only the addition ofthe sterile liquid carrier, for example water for injection, immediatelyprior to use. Extemporaneous injection solutions and suspensions areprepared from sterile powders, granules and tablets of the kindpreviously described. Preferred unit dosage pharmaceutical compositionsare those containing a daily dose or unit daily sub-dose, as hereinabove recited, or an appropriate fraction thereof, of the activeingredient.

It should be understood that in addition to the ingredients particularlymentioned above the pharmaceutical compositions of this invention mayinclude other agents conventional in the art having regard to the typeof pharmaceutical composition in question, for example those suitablefor oral administration may include flavoring agents.

The invention further provides veterinary compositions comprising atleast one active ingredient as above defined together with a veterinarycarrier therefor.

Veterinary carriers are materials useful for the purpose ofadministering the composition and may be solid, liquid or gaseousmaterials which are otherwise inert or acceptable in the veterinary artand are compatible with the active ingredient. These veterinarycompositions may be administered orally, parenterally or by any otherdesired route.

Compounds of the invention may be used to provide controlled releasepharmaceutical formulations containing as an active ingredient one ormore compounds of the invention (“controlled release formulations”) inwhich the release of the active ingredient are controlled and regulatedto allow less frequency dosing or to improve the pharmacokinetic ortoxicity profile of a given active ingredient.

In another embodiment, the present application discloses pharmaceuticalcompositions comprising a compound of the present invention, or apharmaceutically acceptable salt, solvate, or ester thereof, incombination with at least one additional therapeutic agent, and apharmaceutically acceptable carrier or exipient.

For the treatment of Orthomyxoviridae virus infections, preferably, theadditional therapeutic agent is active against Orthomyxoviridae virusinfections, particularly Influenza virus infections. Non-limitingexamples of these active therapeutic agents are viral haemagglutinininhibitors, viral neuramidase inhibitors, M2 ion channel blockers,Orthomyxoviridae RNA-dependent RNA polymerases inhibitors andsialidases. Non-limiting examples of neuramidase inhibitors includeoseltamivir, zanamivir, laninamivir, peramivir and CS-8958. Non-limitingexamples of viral M2 channel inhibitors include amantadine andrimantadine. Non-limiting examples of Orthomyxoviridae RNA-dependent RNApolymerases inhibitors are ribavirin and favipiravir. A non-limitingexample of sialidases is DAS 181. In another embodiment, the additionaltherapeutic agent is selected from the group consisting of ribavirin,oseltamivir, zanamivir, laninamivir, peramivir, amantadine, rimantadine,CS-8958, favipiravir, AVI-7100, alpha-1 protease inhibitor and DAS181.

Many of the infections of the Orthomyxoviridae viruses are respiratoryinfections. Therefore, additional active therapeutics used to treatrespiratory symptoms and sequelae of infection may be used incombination with the compounds of Formula I, II or III. For example,other preferred additional therapeutic agents in combination with thecompounds of Formula I, II or III for the treatment of viral respiratoryinfections include, but are not limited to, bronchodilators andcorticosteroids.

Glucocorticoids, which were first introduced as an asthma therapy in1950 (Carryer, Journal of Allergy, 21, 282-287, 1950), remain the mostpotent and consistently effective therapy for this disease, althoughtheir mechanism of action is not yet fully understood (Morris, J.Allergy Clin. Immunol., 75 (1 Pt) 1-13, 1985). Unfortunately, oralglucocorticoid therapies are associated with profound undesirable sideeffects such as truncal obesity, hypertension, glaucoma, glucoseintolerance, acceleration of cataract formation, bone mineral loss, andpsychological effects, all of which limit their use as long-termtherapeutic agents (Goodman and Gilman, 10th edition, 2001). A solutionto systemic side effects is to deliver steroid drugs directly to thesite of inflammation. Inhaled corticosteroids (ICS) have been developedto mitigate the severe adverse effects of oral steroids. Non-limitingexamples of corticosteroids that may be used in combinations with thecompounds of Formula I are dexamethasone, dexamethasone sodiumphosphate, fluorometholone, fluorometholone acetate, loteprednol,loteprednol etabonate, hydrocortisone, prednisolone, fludrocortisones,triamcinolone, triamcinolone acetonide, betamethasone, beclomethasonediproprionate, methylprednisolone, fluocinolone, fluocinolone acetonide,flunisolide, fluocortin-21-butylate, flumethasone, flumetasone pivalate,budesonide, halobetasol propionate, mometasone furoate, fluticasonepropionate, ciclesonide; or a pharmaceutically acceptable salts thereof.

Other anti-inflamatory agents working through anti-inflamatory cascademechanisms are also useful as additional therapeutic agents incombination with the compounds of Formula I for the treatment of viralrespiratory infections. Applying “anti-inflammatory signal transductionmodulators” (referred to in this text as AISTM), like phosphodiesteraseinhibitors (e.g. PDE-4, PDE-5, or PDE-7 specific), transcription factorinhibitors (e.g. blocking NFκB through IKK inhibition), or kinaseinhibitors (e.g. blocking P38 MAP, JNK, PI3K, EGFR or Syk) is a logicalapproach to switching off inflammation as these small molecules target alimited number of common intracellular pathways—those signaltransduction pathways that are critical points for the anti-inflammatorytherapeutic intervention (see review by P. J. Barnes, 2006). Thesenon-limiting additional therapeutic agents include:5-(2,4-Difluoro-phenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid(2-dimethylamino-ethyl)-amide (P38 Map kinase inhibitor ARRY-797);3-Cyclopropylmethoxy-N-(3,5-dichloro-pyridin-4-yl)-4-difluorormethoxy-benzamide(PDE-4 inhibitor Roflumilast);4-[2-(3-cyclopentyloxy-4-methoxyphenyl)-2-phenyl-ethyl]-pyridine (PDE-4inhibitor CDP-840);N-(3,5-dichloro-4-pyridinyl)-4-(difluoromethoxy)-8-[(methylsulfonyl)amino]-1-dibenzofurancarboxamide(PDE-4 inhibitor Oglemilast);N-(3,5-Dichloro-pyridin-4-yl)-2-[1-(4-fluorobenzyl)-5-hydroxy-1H-indol-3-yl]-2-oxo-acetamide(PDE-4 inhibitor AWD 12-281);8-Methoxy-2-trifluoromethyl-quinoline-5-carboxylic acid(3,5-dichloro-1-oxy-pyridin-4-yl)-amide (PDE-4 inhibitor Sch 351591);4-[5-(4-Fluorophenyl)-2-(4-methanesulfinyl-phenyl)-1H-imidazol-4-yl]-pyridine(P38 inhibitor SB-203850);4-[4-(4-Fluoro-phenyl)-1-(3-phenyl-propyl)-5-pyridin-4-yl-1H-imidazol-2-yl]-but-3-yn-1-ol(P38 inhibitor RWJ-67657);4-Cyano-4-(3-cyclopentyloxy-4-methoxy-phenyl)-cyclohexanecarboxylic acid2-diethylamino-ethyl ester (2-diethyl-ethyl ester prodrug of Cilomilast,PDE-4 inhibitor);(3-Chloro-4-fluorophenyl)-[7-methoxy-6-(3-morpholin-4-yl-propoxy)-quinazolin-4-yl]-amine(Gefitinib, EGFR inhibitor); and4-(4-Methyl-piperazin-1-ylmethyl)-N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-benzamide(Imatinib, EGFR inhibitor).

Agents that inhibit migration of pro-inflammatory cells to the site ofinfection are also useful as additional therapeutic agents incombination with the compounds of Formula I for the treatment of viralrespiratory infections. Non-limiting examples of such agents that actthrough this mechanism and have demonstrated utility in animals by, forexample, reducing the eventual mortality caused by influenza are EV-077(a dual thromboxane synthase inhibitor/thromboxane receptor antagonist)and Fingolimod® (a sphingosine-1-phosphate receptor antagonist).

Combinations comprising inhaled β2-adrenoreceptor agonistbronchodilators such as formoterol, albuterol or salmeterol with thecompounds of Formula I are also suitable, but non-limiting, combinationsuseful for the treatment of respiratory viral infections.

Combinations of inhaled β2-adrenoreceptor agonist bronchodilators suchas formoterol or salmeterol with ICS's are also used to treat both thebronchoconstriction and the inflammation (Symbicort® and Advair®,respectively). The combinations comprising these ICS andβ2-adrenoreceptor agonist combinations along with the compounds ofFormula I are also suitable, but non-limiting, combinations useful forthe treatment of respiratory viral infections.

For the treatment or prophylaxis of pulmonary broncho-constriction,anticholinergics are of potential use and, therefore, useful as anadditional therapeutic agents in combination with the compounds ofFormula I for the treatment of viral respiratory infections. Theseanticholinergics include, but are not limited to, antagonists of themuscarinic receptor (particularly of the M3 subtype) which have showntherapeutic efficacy in man for the control of cholinergic tone in COPD(Witek, 1999);1-{4-Hydroxy-1-[3,3,3-tris-(4-fluoro-phenyl)-propionyl]-pyrrolidine-2-carbonyl}-pyrrolidine-2-carboxylicacid (1-methyl-piperidin-4-ylmethyl)-amide;3-[3-(2-Diethylamino-acetoxy)-2-phenyl-propionyloxy]-8-isopropyl-8-methyl-8-azonia-bicyclo[3.2.1]octane(Ipratropium-N,N-diethylglycinate);1-Cyclohexyl-3,4-dihydro-1H-isoquinoline-2-carboxylic acid1-aza-bicyclo[2.2.2]oct-3-yl ester (Solifenacin);2-Hydroxymethyl-4-methanesulfinyl-2-phenyl-butyric acid1-aza-bicyclo[2.2.2]oct-3-yl ester (Revatropate);2-{1-[2-(2,3-Dihydro-benzofuran-5-yl)-ethyl]-pyrrolidin-3-yl}-2,2-diphenyl-acetamide(Darifenacin); 4-Azepan-1-yl-2,2-diphenyl-butyramide (Buzepide);7-[3-(2-Diethylamino-acetoxy)-2-phenyl-propionyloxy]-9-ethyl-9-methyl-3-oxa-9-azonia-tricyclo[3.3.1.02,4]nonane(Oxitropium-N,N-diethylglycinate);7-[2-(2-Diethylamino-acetoxy)-2,2-di-thiophen-2-yl-acetoxy]-9,9-dimethyl-3-oxa-9-azonia-tricyclo[3.3.1.02,4]nonane(Tiotropium-N,N-diethylglycinate); Dimethylamino-acetic acid2-(3-diisopropylamino-1-phenyl-propyl)-4-methyl-phenyl ester(Tolterodine-N,N-dimethylglycinate);3-[4,4-Bis-(4-fluoro-phenyl)-2-oxo-imidazolidin-1-yl]-1-methyl-1-(2-oxo-2-pyridin-2-yl-ethyl)-pyrrolidinium;1-[1-(3-Fluoro-benzyl)-piperidin-4-yl]-4,4-bis-(4-fluoro-phenyl)-imidazolidin-2-one;1-Cyclooctyl-3-(3-methoxy-1-aza-bicyclo[2.2.2]oct-3-yl)-1-phenyl-prop-2-yn-1-ol;3-[2-(2-Diethylamino-acetoxy)-2,2-di-thiophen-2-yl-acetoxy]-1-(3-phenoxy-propyl)-1-azonia-bicyclo[2.2.2]octane(Aclidinium-N,N-diethylglycinate); or(2-Diethylamino-acetoxy)-di-thiophen-2-yl-acetic acid1-methyl-1-(2-phenoxy-ethyl)-piperidin-4-yl ester.

The compounds of Formula I may also be combined with mucolytic agents totreat both the infection and symptoms of respiratory infections. Anon-limiting example of a mucolytic agent is ambroxol. Similarly, thecompounds of Formula I may be combined with expectorants to treat boththe infection and symptoms of respiratory infections. A non-limitingexample of an expectorant is guaifenesin.

Nebulized hypertonic saline is used to improve immediate and lon-termclearance of small airways in patients with lung diseases (Kuzik, J.Pediatrics 2007, 266). The compounds of Formula I may also be combinedwith nebulized hypertonic saline particularly when the Orthomyxoviridaevirus infection is complicated with bronchiolitis. The combination ofthe compounds of Formula I with hypertonic saline may also comprise anyof the additional agents discussed above. In a preferred aspect,nebulized about 3% hypertonic saline is used.

It is also possible to combine any compound of the invention with one ormore other active therapeutic agents in a unitary dosage form forsimultaneous or sequential administration to a patient. The combinationtherapy may be administered as a simultaneous or sequential regimen.When administered sequentially, the combination may be administered intwo or more administrations.

Co-administration of a compound of the invention with one or more otheractive therapeutic agents generally refers to simultaneous or sequentialadministration of a compound of the invention and one or more otheractive therapeutic agents, such that therapeutically effective amountsof the compound of the invention and one or more other activetherapeutic agents are both present in the body of the patient.

Co-administration includes administration of unit dosages of thecompounds of the invention before or after administration of unitdosages of one or more other active therapeutic agents, for example,administration of the compounds of the invention within seconds,minutes, or hours of the administration of one or more other activetherapeutic agents. For example, a unit dose of a compound of theinvention can be administered first, followed within seconds or minutesby administration of a unit dose of one or more other active therapeuticagents.

Alternatively, a unit dose of one or more additional therapeutic agentscan be administered first, followed by administration of a unit dose ofa compound of the invention within seconds or minutes. In some cases, itmay be desirable to administer a unit dose of a compound of theinvention first, followed, after a period of hours (e.g., 1-12 hours),by administration of a unit dose of one or more other active therapeuticagents. In other cases, it may be desirable to administer a unit dose ofone or more other active therapeutic agents first, followed, after aperiod of hours (e.g., 1-12 hours), by administration of a unit dose ofa compound of the invention.

The third embodiment of the present invention is directed to a methodfor treating an Orthomyxoviridae infection in a mammal in need thereof.The method comprises the step of administering a therapeuticallyeffective amount of a compound of Formula I, as defined above withrespect to the first embodiment of the invention. In a certainembodiment thereof, the compound of Formula I is represented by FormulaII or Formula III, as defined above with respect to the first embodimentof the invention. Terms in the third embodiment of the invention aredefined as above with respect to the first and second embodiments of theinvention. Preferred embodiments of R¹, R², R³, R⁴, R⁵, R⁶ and R⁷ in thethird embodiment of the invention are the same as for the firstembodiment of the invention.

Preferably the compound of Formula I is:

or a pharmaceutically acceptable salt, solvate, or ester thereof, andmore preferably is

or a pharmaceutically acceptable salt, solvate or ester thereof.

In another preferred embodiment of the invention, the compound ofFormula I is:

or a pharmaceutically acceptable salt, solvate, or ester thereof.

In another embodiment of the invention, a therapeutically effectiveamount of a racemate, enantiomer, diastereomer, tautomer, polymorph,pseudopolymorph, amorphous form, or hydrate of a compound of Formula Ior a pharmaceutically acceptable salt, solvate or ester thereof isadministered to a mammal in need thereof.

In another embodiment, provided is the use of a compound of Formula I ora pharmaceutically acceptable salt, solvate, or ester thereof to treat aviral infection caused by an Orthomyxoviridae virus.

In another aspect of this third embodiment, the Orthomyxoviridaeinfection being treated is an Influenza virus A infection. In anotheraspect of this embodiment, the Orthomyxoviridae infection is anInfluenza virus B infection. In another aspect of this embodiment, theOrthomyxoviridae infection is an Influenza virus C infection.

In a preferred embodiment, the method of the invention comprisestreating an Orthomyxoviridae infection in a mammal in need thereof byadministering a therapeutically effective amount of a compound ofFormula I or a pharmaceutically acceptable salt or ester thereof.

In another embodiment, the method of the invention comprises treating anOrthomyxoviridae infection in a mammal in need thereof by administeringa therapeutically effective amount of a pharmaceutical compositioncomprising an effective amount of a Formula I compound, or apharmaceutically acceptable salt, solvate or ester thereof, incombination with a pharmaceutically acceptable diluent or carrier. Anycarrier or diluent known in the art for use in pharmaceuticalcompositions, which is also compatible with the other ingredients of theformulation and physiologically innocuous to the recipient thereof, maybe used in the present invention. Suitable diluents include, but are notlimited to, calcium or sodium carbonate, lactose, calcium or sodiumphosphate.

In another embodiment, the method of the invention comprises treating anOrthomyxoviridae infection in a mammal in need thereof by administeringa therapeutically effective amount of a pharmaceutical compositioncomprising an effective amount of a Formula I compound, or apharmaceutically acceptable salt or ester thereof, in combination withat least one additional therapeutic agent. The additional therapeuticagent may be any therapeutic agent suitable for use with the Formula Icompound. For example, the therapeutic agent may be selected from thegroup consisting of viral haemagglutinin inhibitors, viral neuramidaseinhibitors, M2 ion channel blockers, Orthomyxoviridae RNA-dependent RNApolymerases inhibitors, sialidases, and other drugs for treatingOrthomyxoviridae infections.

In still yet another embodiment, the present invention provides formethods of treating Orthomyxoviridae infections in a patient,comprising: administering to the patient a therapeutically effectiveamount of a compound of Formula I, II, or a pharmaceutically acceptablesalt, solvate, or ester thereof.

In still yet another embodiment, the present application provides formethods of treating Orthomyxoviridae infections in a patient,comprising: administering to the patient a therapeutically effectiveamount of a compound of Formula I, II, or a pharmaceutically acceptablesalt, solvate, or ester thereof, and at least one additional activetherapeutic agent, whereby Orthomyxoviridae polymerase is inhibited.

In still yet another embodiment, the present application provides formethods of treating Orthomyxoviridae infections in a patient,comprising: administering to the patient a therapeutically effectiveamount of a compound of Formula I, II, or a pharmaceutically acceptablesalt, solvate, or ester thereof, and at least one additional activetherapeutic agent selected from the group consisting of interferons,ribavarin analogs, a viral haemagglutinin inhibitor, a viral neuramidaseinhibitor, an M2 ion channel blocker, an Orthomyxoviridae RNA-dependentRNA polymerases inhibitor, a sialidase, and other drugs for treatingOrthomyxoviridae infections.

In still yet another embodiment, the present application provides forthe use of a compound of the present invention, or a pharmaceuticallyacceptable salt, solvate, and/or ester thereof, for the preparation of amedicament for treating an Orthomyxoviridae infections in a patient.

In another aspect of the invention, processes are disclosed below, whichmay be used for preparing Formula I compounds of the invention.

Another aspect of the invention relates to methods of inhibiting theactivity of Orthomyxoviridae polymerase comprising the step of treatinga sample suspected of containing Orthomyxoviridae virus with acomposition of the invention.

Compositions of the invention may act as inhibitors of Orthomyxoviridaepolymerase, as intermediates for such inhibitors, or have otherutilities as described below. The inhibitors will bind to locations onthe surface or in a cavity of Orthomyxoviridae polymerase having ageometry unique to Orthomyxoviridae polymerase. Compositions bindingOrthomyxoviridae polymerase may bind with varying degrees ofreversibility. Those compounds that bind substantially irreversibly areideal candidates for use in this method of the invention. Once labeled,the substantially irreversibly binding compositions are useful as probesfor the detection of Orthomyxoviridae polymerase. Accordingly, theinvention relates to methods of detecting Orthomyxoviridae polymerase ina sample suspected of containing Orthomyxoviridae polymerase comprisingthe steps of: treating a sample suspected of containing Orthomyxoviridaepolymerase with a composition comprising a compound of the inventionbound to a label; and observing the effect of the sample on the activityof the label. Suitable labels are well known in the diagnostics fieldand include stable free radicals, fluorophores, radioisotopes, enzymes,chemiluminescent groups and chromogens. The compounds herein are labeledin conventional fashion using functional groups such as hydroxyl,carboxyl, sulfhydryl or amino.

Within the context of the invention, samples suspected of containingOrthomyxoviridae polymerase include natural or man-made materials suchas living organisms; tissue or cell cultures; biological samples such asbiological material samples (blood, serum, urine, cerebrospinal fluid,tears, sputum, saliva, tissue samples, and the like); laboratorysamples; food, water, or air samples; bioproduct samples such asextracts of cells, particularly recombinant cells synthesizing a desiredglycoprotein; and the like. Typically, the sample will be suspected ofcontaining an organism which produces Orthomyxoviridae polymerase,frequently a pathogenic organism such as Orthomyxoviridae virus. Samplescan be contained in any medium including water and organic solvent\watermixtures. Samples include living organisms such as humans, and man madematerials such as cell cultures.

The treating step of the invention comprises adding the composition ofthe invention to the sample or adding a precursor of the composition tothe sample. The addition step comprises any method of administration asdescribed herein.

If desired, the activity of Orthomyxoviridae polymerase afterapplication of the composition can be observed by any method includingdirect and indirect methods of detecting Orthomyxoviridae polymeraseactivity. Quantitative, qualitative, and semiquantitative methods ofdetermining Orthomyxoviridae polymerase activity are all contemplated.Typically one of the screening methods described above are applied,however, any other method such as observation of the physiologicalproperties of a living organism are also applicable.

Organisms that contain Orthomyxoviridae polymerase include theOrthomyxoviridae virus. The compounds of this invention are useful inthe treatment or prophylaxis of Orthomyxoviridae infections in animalsor in man.

In still yet another embodiment, the present application provides formethods of inhibiting Orthomyxoviridae RNA-dependent RNA polymerase in acell, comprising: contacting a cell infected with Orthomyxoviridae viruswith an effective amount of a compound of Formula I, II, or apharmaceutically acceptable salt, solvate, or ester thereof, whereby theOrthomyxoviridae polymerase is inhibited. IN an aspect of thisembodiment, the cell is also contacted by at least one additionaltherapeutic agent. In certain embodiments of the invention, theOrthomyxoviridae RNA-dependent RNA polymerase may be a Influenza virus ARNA-dependent RNA polymerase, a Influenza virus B RNA-dependent RNApolymerase, a Influenza virus C RNA-dependent RNA polymerase, ormixtures thereof.

In still yet another embodiment, the present application provides formethods of inhibiting Orthomyxoviridae polymerase in a cell, comprising:contacting a cell infected with Orthomyxoviridae virus with an effectiveamount of a compound of Formula I, II, or a pharmaceutically acceptablesalt, solvate, or ester thereof, and at least one additional activetherapeutic agent selected from the group consisting of interferons,ribavirin analogs, viral neuramidase inhibitors, viral neuramidaseinhibitors, M2 ion channel blockers, Orthomyxoviridae RNA-dependent RNApolymerases inhibitors, sialidases and other drugs used to treatOrthomyxoviridae virus infections.

In another aspect, the invention also provides processes and novelintermediates disclosed herein which are useful for preparing Formula Icompounds of the invention.

In other aspects, novel methods for synthesis, analysis, separation,isolation, purification, characterization, and testing of the compoundsof this invention are provided.

Also falling within the scope of this invention are the in vivometabolic products of the compounds described herein, to the extent suchproducts are novel and unobvious over the prior art. Such products mayresult for example from the oxidation, reduction, hydrolysis, amidation,esterification and the like of the administered compound, primarily dueto enzymatic processes. Accordingly, the invention includes novel andunobvious compounds produced by a process comprising contacting acompound of this invention with a mammal for a period of time sufficientto yield a metabolic product thereof. Such products typically areidentified by preparing a radiolabelled (e.g. ¹⁴C or ³H) compound of theinvention, administering it parenterally in a detectable dose (e.g.greater than about 0.5 mg/kg) to an animal such as rat, mouse, guineapig, monkey, or to man, allowing sufficient time for metabolism to occur(typically about 30 seconds to 30 hours) and isolating its conversionproducts from the urine, blood or other biological samples. Theseproducts are easily isolated since they are labeled (others are isolatedby the use of antibodies capable of binding epitopes surviving in themetabolite). The metabolite structures are determined in conventionalfashion, e.g., by MS or NMR analysis. In general, analysis ofmetabolites is done in the same way as conventional drug metabolismstudies well-known to those skilled in the art. The conversion products,so long as they are not otherwise found in vivo, are useful indiagnostic assays for therapeutic dosing of the compounds of theinvention even if they possess no Orthomyxoviridae polymerase inhibitoryactivity of their own.

Recipes and methods for determining stability of compounds in surrogategastrointestinal secretions are known. Compounds are defined herein asstable in the gastrointestinal tract where less than about 50 molepercent of the protected groups are deprotected in surrogate intestinalor gastric juice upon incubation for 1 hour at 37° C. Simply because thecompounds are stable to the gastrointestinal tract does not mean thatthey cannot be hydrolyzed in vivo. The prodrugs of the inventiontypically will be stable in the digestive system but may besubstantially hydrolyzed to the parental drug in the digestive lumen,liver or other metabolic organ, or within cells in general.

EXAMPLES

Certain abbreviations and acronyms are used in describing theexperimental details. Although most of these would be understood by oneskilled in the art, Table 1 contains a list of many of theseabbreviations and acronyms.

Table 1. List of abbreviations and acronyms.

Abbreviation Meaning Bn Benzyl DMSO Dimethylsulfoxide DMFDimethylformamide MCPBA meta-chloroperbenzoic acid m/z mass to chargeratio MS or ms mass spectrum THF tetrahydrofuran δ parts per millionreferenced to residual non-deuterated solvent peak

Preparation of Compounds Compound 1:(2S,3R,4R,5R)-4-(benzyloxy)-5-(benzyloxymethyl)-2-(2,4-bis(methylthio)imidazo[1,2-f][1,2,4]triazin-7-yl)-3-fluorotetrahydrofuran-2-01

To a mixture of 7-bromo-2,4-bis(methylthio)imidazo[1,2-f][1,2,4]triazine(2.5 g, 7.57 mmol) in THF (30 ml) at −78° C. was dropwise added nBuLi(1.6 M in hexane, 6.15 ml, 9.84 mmol). After stirring at −78° C. for 30minutes,(3R,4R,5R)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorodihydrofuran-2(3H)-one(2.43 g, 8.33 mmol) in THF (5 ml) was dropwise added. After stirring at−78° C. for 3 hours, the mixture was allowed to warm to roomtemperature. The mixture was then stirred at room temperature for 30minutes and then quenched with saturated NH₄Cl. The reaction wasextracted with ethyl acetate. The layers were separated and the combinedorganic layers were washed with brine, dried over Na₂SO₄ andconcentrated to provide crude, which was purified by flash columnchromatography with ethyl acetate/hexanes to provide the desiredcompound(2S,3R,4R,5R)-4-(benzyloxy)-5-(benzyloxymethyl)-2-(2,4-bis(methylthio)imidazo[1,2-f][1,2,4]triazin-7-yl)-3-fluorotetrahydrofuran-2-ol(1) (2 g, 48%) as yellow foam. MS (m/z): 543.2 [M+H]⁺.

Compound 2:7-((2S,3S,4R,5R)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorotetrahydrofuran-2-yl)-2,4-bis(methylthio)imidazo[1,2-f][1,2,4]-triazine

To a solution of(2S,3R,4R,5R)-4-(benzyloxy)-5-(benzyloxymethyl)-2-(2,4-bis(methylthio)imidazo[1,2-f][1,2,4]triazin-7-yl)-3-fluorotetrahydrofuran-2-ol(1) (300 mg, 0.55 mmol) in dichloromethane (3 ml) at −78° C. was addedBF₃.OEt₂ (1.20 ml, 8.81 mmol) dropwise, followed by addition of Et₃SiH(1.52 ml, 8.8 1 mmol). The reaction was allowed warm to room temperatureand stirred for 2 hours and then quenched with saturated NaHCO₃ and thenextracted with dichloromethane. The organic layers were separated,washed with brine, dried over Na₂SO₄ and concentrated to provide crude,which was purified by flash column chromatography with ethylacetate/hexanes to provide the desired compound7-((2S,3S,4R,5R)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorotetrahydrofuran-2-yl)-2,4-bis(methylthio)imidazo[1,2-f][1,2,4]triazine(2) (218 mg, 75%). MS (m/z): 527.2 [M+H]⁺.

Compound 3:7-((2S,3S,4R,5R)-4-(benzyloxy)-5-(benzyloxymethy)-3-fluorotetrahydrofuran-2-yl)-2-(methylthio)imidazo[1,2-f][1,2,4]-triazin-4-amine

7-((2S,3S,4R,5R)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorotetrahydrofuran-2-yl)-2,4-bis(methylthio)imidazo[1,2-f][1,2,4]triazine(2) (390 mg, 0.74 mmol) in liquid ammonia (120 ml) was heated at 60° C.in a steel bomb for 18 hours. The bomb was cooled down to roomtemperature and the reaction was purified by flash column chromatographywith ethyl acetate/hexanes to provide the desired compound7-((2S,3S,4R,5R)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorotetrahydrofuran-2-yl)-2-(methylthio)imidazo[1,2-f][1,2,4]triazin-4-amine(3) (330 mg, 89%). MS (m/z): 496.2 [M+H]⁺

Compound 4:7-((2S,3S,4R,5R)-4-(benzyloxy)-5-(benzyloxymethy)-3-fluorotetrahydrofuran-2-yl)-2-(methylsulfonyl)imidazo[1,2-f][1,2,4]triazin-4-amine

To a solution of7-((2S,3S,4R,5R)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorotetrahydrofuran-2-yl)-2-(methylthio)imidazo[1,2-f][1,2,4]triazin-4-amine(3) (300 mg, 0.57 mmol) in dichloromethane (10 ml) at 0° C. was added3-chloroperbenzoic acid (MCPBA, 77%) (627 mg, 3.42 mmol) in one portion.The reaction was allowed to warm to room temperature and stirred for 2hours. The reaction was quenched with a 20% NaS₂O₃ solution in H₂O (15ml) and allowed to stir for 20 minutes. The layers were separated, andthe aqueous solution was extracted with dichloromethane. The combinedorganic layers were washed with saturated NaHCO₃ and brine, and thendried over Na₂SO₄ and concentrated to provide a crude mixture which wasfurther purified by silica gel column chromatography with ethylacetate/dichloromethane to provide the desired product7-((2S,3S,4R,5R)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorotetrahydrofuran-2-yl)-2-(methylsulfonyl)imidazo[1,2-f][1,2,4]triazin-4-amine(4) (276 mg, 87%) as clear oil. MS (m/z): 528.1 [M+H]⁺.

Compound 5:7-((2S,3S,4R,5R)-4-(benzyloxy)-5-(benzyloxymethy)-3-fluorotetrahydrofuran-2-yl)imidazo[1,2-f][1,2,4]-triazine-2,4-diamine

7-((2S,3S,4R,5R)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorotetrahydrofuran-2-yl)-2-(methylsulfonyl)imidazo[1,2-f][1,2,4]triazin-4-amine(4) (276 mg, 0.52 mmol) in liquid ammonia (100 ml) was heated at 110° C.for 26 hours in a steel bomb. The bomb was cooled down to roomtemperature, and the crude reaction was purified by flash columnchromatography with ethyl acetate/dichloromethane to provide the desiredcompound7-((2S,3S,4R,5R)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorotetrahydrofuran-2-yl)imidazo[1,2-f][1,2,4]triazine-2,4-diamine(5) (185 mg, 78%). MS (m/z): 465.3 [M+H]⁺

Compound 6:(2S,3S,4R,5R)-5-(2,4-diaminoimidazo[1,2-f][1,2,4]-triazin-7-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol

To a solution of7-((2S,3S,4R,5R)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorotetrahydrofuran-2-yl)imidazo[1,2-f][1,2,4]triazine-2,4-diamine(5) (145 mg, 0.31 mmol) in acetic acid (10 ml) was added 10% Pd/CDegussa type E101 NE/W (290 mg). The reaction atmosphere was exchangedfor H₂ (g) and the reaction stirred for 18 hours. The catalyst wasremoved by filtration and the mixture concentrated under reducedpressure. The crude was dried to provide the desired product(2S,3S,4R,5R)-5-(2,4-diaminoimidazo[1,2-f][1,2,4]triazin-7-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol(6) (85 mg, 96%) as a white solid. MS (m/z): 285.2 [M+H]⁺.

¹H NMR (400 MHz, CD₃OD): δ 7.45 (s, 1H), 5.44-5.38 (m, 1H), 5.24-5.11(d, J=, 1H), 4.38-4.33 (m, 1H), 3.98 (s, 1H), 3.91-3.70 (m 2H).

¹⁹F (376 MHz, CD₃OD): δ (−199.86)-(−200.13) (m)

Compound 7:2-amino-7-(2S,3S,4R,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)imidazo[1,2-f][1,2,4]-triazin-4(3H)-one

To a solution of(2S,3S,4R,5R)-5-(2,4-diaminoimidazo[1,2-f][1,2,4]triazin-7-yl)-4-fluoro-2-(hydroxymethytetrahydrofuran-3-ol(6) (310 mg, 1.09 mmol) in 800 ml water was added bovine spleenadenosine deaminase type IX (Cas No. 9026-93-1, 205 μL). The solutionwas placed in a 37° C. water bath for 16 hours. The solution wasconcentrated and the final compound was crystallized separate fromimpurities using water as the crystallizing solvent. Solids werecollected and dried to provide2-amino-7-(2S,3S,4R,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)imidazo[1,2-f][1,2,4]triazin-4(3H)-one(7) (246 mg, 80%) as a pure, off-white solid. MS (m/z): 286.2 [M+H]⁺.

¹H NMR (400 MHz, DMSO-d₆): δ 11.27 (s, 1H), 7.41 (s, 1H), 6.24 (s, 2H),5.43-5.42 (m, 1H), 5.26-5.20 (d, J=22.8 Hz, 1H), 5.09-4.85 (m, 2H),4.14-4.09 (m, 1H), 3.77 (s, 1H), 3.69-3.51 (m, 2H).

¹⁹F (376 MHz, DMSO-d₆): 8 (−196.68)-(−196.94) (m)

Compound 8:((2R,3R,4R,5S)-5-(2-amino-4-oxo-3,4-dihydroimidazo[1,2-f][1,2,4]-triazin-7-yl)-4-fluoro-3-hydroxy-3-hydroxytetrahydrofuran-2-yl)methyltetrahydrogen triphosphate

2-amino-7-(2S,3S,4R,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)imidazo[1,2-f][1,2,4]triazin-4(3H)-one(7) (12 mg, 0.042 mmol) was dissolved in trimethylphosphate (1 mL) underan inert atmosphere (N₂). Phosphorous oxychloride (58 mg, 0.378 mmol)was added and the mixture was stirred at 0° C. for 2 hours and at roomtemperature for 2 hours. Monitoring by analytical ion-exchange columndetermined the time at which >80% of monophosphate was formed. Thesolution was cooled to 0° C., and a solution of tributylamine (0.15 mL,0.63 mmol) and triethylammonium pyrophosphate (0.25 g, 0.55 mmol) inanhydrous DMF (1 mL) was added. The reaction mixture was stirred at 0°C. for 2.5 hours and then quenched by the addition of 1Ntriethylammonium bicarbonate solution in H₂O (6 mL). The mixture wasconcentrated under reduced pressure and the residue re-dissolved in H₂O.The solution was subjected to ion exchange chromatography to yield thedesired product((2R,3R,4R,5S)-5-(2-amino-4-oxo-3,4-dihydroimidazo[1,2-f][1,2,4]triazin-7-yl)-4-fluoro-3-hydroxy-3-hydroxytetrahydrofuran-2-yl)methyltetrahydrogen triphosphate (8) (as the tetratriethylammonium salt) (11mg, 28% yield). MS (m/z): 526.0 [M+H]⁺.

¹H NMR (400 MHz, D₂O): δ 7.53 (s, 1H), 5.43-5.37 (d, J=24.8 Hz, 1H),5.29-5.15 (d, J=55.2, 1H), 4.52-3.47 (m, 4H).

¹⁹F (376 MHz, D₂O): δ (−197.33)-(−197.60) (m, 1F)

³¹P (162 MHz, D₂O) δ (−10.66)+10.78) (d, J=48.4 Hz, 1P),(−11.070)-(−11.193) (d, J=49.2 Hz, 1P), (−22.990)-(−23.236) (m, 1P).

HPLC ion exchange: Solvent A: Water; Solvent B: 1M triethylammoniumbicarbonate.

0-50% over 12 minutes, then 100% for 5 minutes, then back to 0% in 5minutes.

Column: Dionex, DNAPac PA-100, 4×250 mm.

T_(R)=12.04 min

Compound 9:7-((2S,3S,4R,5R)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorotetrahydrofuran-2-yl)imidazo[1,2-f][1,2,4]-triazine-4-amine

To a solution of7-((2S,3S,4R,5R)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorotetrahydrofuran-2-yl)-2-(methylsulfonyl)imidazo[1,2-f][1,2,4]triazin-4-amine(4) (63 mg, 0.12 mmol) in THF (5 ml) at −78° C. was added LiBHEt₃ (1.0 Min THF, 4.78 ml, 4.78 mmol) drop-wise. The reaction was warmed to roomtemperature and stirred at room temperature for 31 hours. The reactionmixture was quenched with ice water and extracted with ethyl acetate.The organic solution was washed with brine and concentrated to give acrude mixture which was dissolved in CH₃OH and concentrated in vacuo(3×). The crude was purified by silica gel chromatography with ethylacetate/dichloromethane to provide the desired product7-((2S,3S,4R,5R)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorotetrahydrofuran-2-yl)imidazo[1,2-f][1,2,4]triazine-4-amine(9) (50 mg, 95% yield). MS (m/z): [M+H]⁺450.3.

Compound 10:(2S,3S,4R,5R)-5-(4-aminoimidazo[1,2-f][1,2,4]-triazin-7-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol

To a solution of7-((2S,3S,4R,5R)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorotetrahydrofuran-2-yl)imidazo[1,2-f][1,2,4]triazine-4-amine(9) (50 mg, 0.11 mmol) in acetic acid (5 ml) was added 10% Pd/C (100mg). The reaction vessels atmosphere was exchanged for hydrogen and thereaction was stirred at room temperature overnight. The reaction wasfiltered through celite and washing with CH₃OH. The filtrate wasconcentrated to give a crude mixture which was purified by silica gelcolumn chromatography using CH₃OH/dichloromethane to provide the desiredproduct(2S,3S,4R,5R)-5-(4-aminoimidazo[1,2-f][1,2,4]triazin-7-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol(10) as a white solid (23 mg, 77% yield). MS (m/z): 270.2 [M+H]⁺.

¹H NMR (400 MHz, DMSO-d₆): δ 8.22 (d, J=26 Hz, 2H), 8.07 (s, 1H), 7.67(s, 1H), 5.50-5.48 (d, J=6.4, 1H), 5.42-5.36 (m, 1H), 5.19-5.03 (m, 1H),4.88-4.85 (m, 1H), 4.19-4.11 (m, 1H), 3.83-3.81 (m, 1H), 3.72-3.67 (m,1H), 3.54-3.50 (m, 1H).

¹⁹F (376 MHz, CD₃OD): δ (−196.69)-(−196.95) (m)

Compound 11:7-((2S,3S,4R,5R)-4-(benzyloxy)-5-(benzyloxymethy)-3-fluorotetrahydrofuran-2-yl)-2-fluoroimidazo[1,2-f][1,2,4]-triazin-4-amine

7-((2S,3S,4R,5R)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorotetrahydrofuran-2-yl)imidazo[1,2-f][1,2,4]triazine-2,4-diamine(5) (140 mg, 0.30 mmol) in 4 mL 50% HF/pyridine was stirred in a −10° C.bath, and 45 μL (0.38 mmol) t-butyl nitrite was added. The reaction wasstirred at low temperature for 1 hour. The reaction was quenched byaddition of 50 mL of H₂O and the aqueous layer extracted 2×50 mLdichloromethane. The combined organics were dried over Na₂SO₄ andconcentrated. The residue chromatographed on 6 g silica gel to providethe desired compound7-((2S,3S,4R,5R)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorotetrahydrofuran-2-yl)-2-fluoroimidazo[1,2-f][1,2,4]triazin-4-amine(11) (50 mg, 36%). MS (m/z): 468.2 [M+H]⁺.

¹H NMR (400 MHz, CDCl₃): 7.60 (s, 1H), 7.3-7.2 (bm, 10H), 7.14 (bs, 1H),6.37 (bs, 1H), 5.46-5.48 (dd, J=23.2, 2.4 Hz, 1H), 5.29-5.15 (m, 1H),4.72 (m, 1H), 4.56-4.52 (m, 2H), 4.29 (m, 2H), 3.83-3.81 (m, 1H),3.65-3.63 (m, 1H).

¹⁹F (376 MHz, CDCl₃): δ −69.1 (s), (−197.7)-(−198.0) (m).

Compound 12:(2R,3R,4R,5S)-5-(4-amino-2-fluoroimidazo[1,2-f][1,2,4]-triazin-7-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol

To a solution of7-((2S,3S,4R,5R)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorotetrahydrofuran-2-yl)imidazo[1,2-f][1,2,4]triazine-4-amine(11) (50 mg, 0.11 mmol) in acetic acid (8 ml), 10% Pd/C (100 mg) wasadded. The reaction vessel's atmosphere was exchanged for hydrogen andthe reaction was stirred at room temperature overnight. The reaction wasfiltered through celite and washed with acetic acid and then CH₃OH. Thefiltrate was concentrated to give a crude mixture that was purified byreversed phase HPLC to provide the desired product(2R,3R,4R,5S)-5-(4-amino-2-fluoroimidazo[1,2-f][1,2,4]triazin-7-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol(12) as a white solid (26 mg, 84%). MS (m/z): 288.1 [M+H]⁺.

¹H NMR (400 MHz, CD₃OD): δ 7.66 (s, 1H), 5.47-5.41 (dd, J=23.6, 2.4 Hz,1H), 5.21-5.06 (m, 1H), 4.35-4.27 (m, 1H), 3.93 (bm, 1H), 3.88 (m, 1H),3.68 (m, 1H).

¹⁹F (376 MHz, CD₃OD): 8-72.15 (s), (−199.39)-(−196.69) (m)

Compound 13:7-bromo-4-ethoxy-2-(methylthio)imidazo[1,2-f][1,2,4]-triazine

To a mixture of 7-bromo-2,4-bis(methylthio)imidazo[1,2-f][1,2,4]triazine(1.0 g, 3.45 mmol) in EtOH (25 ml) at room temperature, NaOEt (21% inEtOH, 1.28 ml, 3.45 mmol) was added. After stirring at room temperaturefor 1 hour, the reaction was quenched with AcOH (1 mL). The solventswere removed under reduced pressure, and the mixture was partitionedbetween CH₂Cl₂ and Y2 saturated brine. The organics were separated,dried over Na₂SO₄, solids removed by filtration and the solvent removedunder reduced pressure. The crude material was purified by flash columnchromatography with ethyl acetate/hexanes to provide the desiredcompound 7-bromo-4-ethoxy-2-(methylthio)imidazo[1,2-f][1,2,4]tiazine(13) (791 mg, 79%) as a yellow foam. MS (m/z): 288.9/290.8 [M+H]⁺.

Compound 14:(2S,3R,4R,5R)-4-(benzyloxy)-5-(benzyloxymethy)-2-(4-ethoxy-2-(methylthio)imidazo[1,2-f][1,2,4]-triazin-7-yl)-3-fluorotetrahydrofuran-2-ol

To a mixture of7-bromo-4-ethoxy-2-(methylthio)imidazo[1,2-f][1,2,4]triazine (13) (917mg, 3.17 mmol) in THF (15 ml) at −78° C. was added LaCl₃*2LiCl (0.6M inTHF, 5.28 mL, 3.17 mmol) followed by the dropwise addition of nBuLi (2.5M in hexane, 1.27 ml, 3.17 mmol). After stirring at −78° C. for 30minutes,(3R,4R,5R)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorodihydrofuran-2(3H)-one(805 mg, 2.44 mmol) in THF (10 ml) was dropwise added. After, stirringat −78° C. for 30 min and allowing the mixture to warm to roomtemperature, the mixture was stirred at room temperature for 30 minutesand then quenched with AcOH. The reaction was extracted with ethylacetate. The layers were separated, and the combined organic layers werewashed with brine, dried over Na₂SO₄ and concentrated to provide crude,which was purified by flash column chromatography with ethylacetate/hexanes, to provide the desired compound(2S,3R,4R,5R)-4-(benzyloxy)-5-(benzyloxymethyl)-2-(4-ethoxy-2-(methylthio)imidazo[1,2-f][1,2,4]triazin-7-yl)-3-fluorotetrahydrofuran-2-ol(14) (244 mg, 19%) as yellow foam. MS (m/z): 541.1 [M+H]⁺.

Compound 15:7-((2S,3S,4R,5R)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorotetrahydrofuran-2-yl)-4-ethoxy-2-(methylthio)imidazo[1,2-f][1,2,4]-triazine

To a solution of(2S,3R,4R,5R)-4-(benzyloxy)-5-(benzyloxymethyl)-2-(4-ethoxy-2-(methylthio)imidazo[1,2-f][1,2,4]triazin-7-yl)-3-fluorotetrahydrofuran-2-ol(X) (244 mg, 0.45 mmol) in CH₂Cl₂ (5 ml) at 0° C. was added BF₃.OEt₂(900 μl, 3.5 mmol) drop-wise, followed by addition of Et₃SiH (600 μl,3.5 mmol). The reaction was allowed to warm to room temperature andstirred for 3 hours. The reaction was quenched with saturated NaHCO₃ andextracted with CH₂Cl₂. The organic layers were separated, washed withbrine, dried over Na₂SO₄ and concentrated to give crude, which waspurified by flash column chromatography with ethyl acetate/hexanes, toprovide the desired compound7-((2S,3S,4R,5R)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorotetrahydrofuran-2-yl)-4-ethoxy-2-(methylthio)imidazo[1,2-f][1,2,4]triazine(15) (107 mg, 46%). MS (m/z): 525.1 [M+H]⁺.

Compound 16:7-((2S,3S,4R,5R)-4-(benzyloxy)-5-(benzyloxymethy)-3-fluorotetrahydrofuran-2-yl)-4-ethoxy-2-(methylsulfonyl)imidazol[1,2-f][1,2,4]-triazine

To a solution of7-((2S,3S,4R,5R)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorotetrahydrofuran-2-yl)-4-ethoxy-2-(methylthio)imidazo[1,2-f][1,2,4]triazin(15) (107 mg, 0.204 mmol) in CH₂Cl₂ (3 ml) at room temperature was added3-chloroperbenzoic acid (MCPBA, 77%) (100 mg, 0.443 mmol) in oneportion. The reaction was stirred at room temperature for 4 hours. Thereaction was quenched with a 20% NaS₂O₃ solution in H₂O (5 ml) andallowed to stir for 20 minutes. The layers were separated and theaqueous solution was extracted with CH₂Cl₂. The combined organic layerswere washed with saturated NaHCO₃, brine, dried over Na₂SO₄ andconcentrated to provide crude7-((2S,3S,4R,5R)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorotetrahydrofuran-2-yl)-4-ethoxy-2-(methylsulfonyl)imidazo[1,2-f][1,2,4]triazine(16), which was carried forward without purification. MS (m/z): 557.1[M+H]⁺.

Compound 17:2-azido-7-((2S,3S,4R,5R)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorotetrahydrofuran-2-yl)-4-ethoxyimidazo[1,2-f][1,2,4]triazine

To a solution of NaN₃ (66 mgs, 1.01 mmol) in DMSO (5 mL) was added7-((2S,3S,4R,5R)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorotetrahydrofuran-2-yl)-4-ethoxy-2-(methylsulfonyl)imidazo[1,2-f][1,2,4]triazine(16) (113 mgs, 0.203 mmol) in one portion. The reaction was allowed tostir at room temperature for 16 hours. The mixture was partitionedbetween EtOAc/H₂O. The organics were separated and dried over Na₂SO₄,and purified by silica gel chromatograghy with EtOAc/hexanes to provide2-azido-7-((2S,3S,4R,5R)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorotetrahydrofuran-2-yl)-4-ethoxyimidazo[1,2-f][1,2,4]triazine(17) (93 mgs, 88%) as an off-white solid. MS (m/z): 520.05 [M+H]⁺.

Compound 18:(2R,3R,4R,5S)-5-(2-amino-4-ethoxyimidazo[1,2-f][1,2,4]-triazine-7-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol

A solution of2-azido-7-((2S,3S,4R,5R)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorotetrahydrofuran-2-yl)-4-ethoxyimidazo[1,2-f][1,2,4]triazine(17) (93 mg, 0.18 mmol) in CH₃OH (5 mL) was purged with argon, and 10%Pd/C (100 mg) was added. The reaction vessel was evacuated and backfilled with H₂ three times. The reaction mixture was then allowed tostir under a hydrogen atmosphere for 16 hours. The solids were filteredoff, and the organics were removed under reduced pressure to give crudematerial that was purified by HPLC to give(2R,3R,4R,5S)-5-(2-amino-4-ethoxyimidazo[1,2-f][1,2,4]triazine-7-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol(18) (27 mg, 48%) as a white solid. MS (m/z): 314.10 [M+H]⁺.

¹H NMR (400 MHz, DMSO-d₆): δ 7.526 (s, 1H), 8.07 (s, 1H), 6.55 (s, 2H),5.43-5.41 (d, J=6.46 Hz, 1H), 5.33-5.27 (dd, J=2.25 and 22.69 Hz, 1H),5.11-4.96 (m, 1H), 4.84 (t, J=5.58 Hz, 1H), 4.52-4.47 (q, J=7.04 Hz,2H), 4.17-4.07 (m, 1H), 3.78-3.76 (m, 1H), 3.69-3.65 (m, 1H), 3.511-3.45(m, 1H), 1.37 (t, J=7.04 Hz, 3H).

¹⁹F (376 MHz, DMSO-d₆): 8 (−196.79)-(−197.05) (m)

Compound 19:(2R,3R,4R,5S)-5-(2-amino-4-methoxyimidazo[1,2-f][1,2,4]triazin-7-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol

(2R,3R,4R,5S)-5-(2-amino-4-methoxyimidazo[1,2-f][1,2,4]triazin-7-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol(19) was prepared in a directly analogous manner as that used for thepreparation of(2R,3R,4R,5S)-5-(2-amino-4-ethoxyimidazo[1,2-f][1,2,4]triazine-7-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol,except NaOMe in MeOH was used instead of NaOEt in EtOH in the first stepof the synthesis. MS (m/z): 300.18 [M+H]⁺.

¹H NMR (400 MHz, CD₃OD): δ 7.56 (s, 1H), 5.46 (dd, J=24, 2.4 Hz, 1H),5.15 (ddd, J=54.8, 4.4, 2.4 Hz, 1H), 4.34 (ddd, J=4.4, 8, 20.4 Hz, 1H),4.15 (s, 3H), 3.97 (m, 1H), 3.91 (dd, J=2.4, 12.4 Hz, 1H), 3.72 (dd,J=4.4, 12 Hz, 1H).

¹⁹F (376 MHz, CD₃OD): 8 (−198.98)-(−199.25) (m).

Anti-Influenza Assays

Influenza RNA Polymerase Inhibition (IC₅₀) Assay

Influenza A/PR/8/34 (H₁N₁) purified virus was obtained from AdvancedBiotechnologies Inc. (Columbia, Md.) as suspension in PBS buffer.Virions were disrupted by exposure to an equal volume of 2% Triton X-100for 30 minutes at room temperature in a buffer containing 100 mMTris-HCl, pH 8, 200 mM KCl, 3 mM dithiothreitol [DTT], 10% glycerol, 10mM MgCl₂, 2 U/mL RNasin Ribonuclease Inhibitor, and 2 mg/mLLysolechithin type V (Sigma, Saint Louis, Mo.). The virus lysate wasstored at −80° C. in aliquots.

The concentrations refer to final concentrations unless mentionedotherwise. Nucleotide analog inhibitors were serially diluted 3 fold inwater and added to reaction mix containing 10% virus lysate (v/v), 100mM Tris-HCl (pH 8.0), 100 mM KCl, 1 mM DTT, 10% glycerol, 0.25%Triton-101 (reduced), 5 mM MgCl₂, 0.4 U/mL RNasin, and 200 μM ApGdinucleotide primer (TriLink, San Diego Calif.). Reactions wereinitiated by addition of ribonucleotide triphosphate (NTP) substrate mixcontaining one α-³³P labeled NTP and 100 μM of the other three naturalNTPs (PerkinElmer, Shelton, Conn.). The radiolabel used for each assaymatched the class of nucleotide analog screened. The concentrations forthe limiting natural NTP are 20, 10, 2, and 1 μM for ATP, CTP, UTP, andGTP respectively. The molar ratio of un-radiolabeled: radiolabeled NTPwere in the range of 100-400:1.

Reactions were incubated at 30° C. for 90 minutes then spotted onto DE81filter paper. Filters were air dried, washed 0.125 M Na₂HPO₄ (3×), water(1×), and EtOH (1×), and air dried before exposed to Typhoon phosphorimager and radioactivity was quantified on a Typhoon Trio (GEHealthcare, Piscataway N.J.). IC₅₀ values were calculated for inhibitorsby fitting the data in GraphPad Prism with a sigmoidal dose responsewith variable slope equation, fixing the Ymax and Ymin values at 100%and 0%. The IC₅₀ for((2R,3R,4R,5S)-5-(2-amino-4-oxo-3,4-dihydroimidazo[1,2-f][1,2,4]triazin-7-yl)-4-fluoro-3-hydroxy-3-hydroxytetrahydrofuran-2-yl)methyltetrahydrogen triphosphate (Compound 8) was determined to be 2.8 μM.

Normal Human Bronchial/Tracheal Epithelial Cell Influenza InfectionAssay (EC₅₀)

Normal Human Bronchial/Tracheal Epithelial Cells (Lonza, BaselSwitzerland) are seeded to 384-well plates at a density of 4000 cellsper well in BEGM medium supplemented with growth factors (Lonza, BaselSwitzerland). Medium is removed next day, and cells are washed threetimes with 100 μL of RPMI+ 1% BSA (RPMI-BSA). 30 μL of RPMI-BSA is addedto cells thereafter. Compounds are 3-fold serially diluted in DMSO, and0.4 μL of compound dilutions are stamped to plates. Influenza A virusHK/8/68 (Advanced Biotechnology Inc, Columbia, Md., 13.5 MOI), PC/1/73(ATCC Manassas, Va., 0.3 MOI) and Influenza B virus B/Lee/40 ((ATCCManassas, Va., 10 MOI) are added to cells in 10 μL of RPMI-BSA mediumsupplemented with 8 ug/mL trypsin (Worthington, Lakewood, N.J.). After afive day incubation, 40 μl, of buffer containing 66 mM Mes pH 6.5, 8 mMCaCl₂, 0.5% NP-40 and 100 μM neuramidase substrate(2′-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic acid sodium salthydrate, Sigma Aldrich, St. Luis, Mo.) is added to cells. Fluorescenceof the product of hydrolysis is read using excitation at 360 nm andemission at 450 nm after a 1 hour incubation at 37° C. EC₅₀ values arecalculated by non-linear regression of multiple data sets.

The following table summarizes EC₅₀s determined by this assay:

Compound Infl A PC/1/73 EC₅₀ Infl B Lee/40 EC₅₀ 19 30 μM 36 μM 18 >200μM  >200 μM  12 >100 μM  >100 μM  10 0.9 μM  0.9 μM  7 27 μM 37 μM 6 21μM 51 μM

While the invention has been described with reference to variousspecific and preferred embodiments and techniques, it will be understoodthat they are not intended to limit the invention to those embodiments.One skilled in the art will understand that many variations andmodifications may be made while remaining within the spirit and scope ofthe invention. The invention is intended to cover all alternatives,modifications, and equivalents, which may be included within the scopeof the present invention.

What is claimed is:
 1. A compound of Formula I:

or a pharmaceutically acceptable salt, solvate, or ester thereof;wherein: each of R¹ and R⁷ is independently H, halogen, OR⁸,(C₁-C₈)haloalkyl, CN, N₃, (C₁-C₈)alkyl, (C₁-C₈)substituted alkyl,(C₂-C₈)alkenyl, (C₂-C₈)substituted alkenyl, (C₂-C₈)alkynyl or(C₂-C₈)substituted alkynyl, wherein the substituent is selected from thegroup consisting of —X, —R^(b), —OH, ═O, —OR^(b), —SR^(b), —S⁻, —NR^(b)₂, —N⁺R^(b) ₃, ═NR^(b), —CX₃, —CN, —OCN, —SCN, —N═C═O, —NCS, —NO, —NO₂,═N₂, —N₃, —NHC(═O)R^(b), —OC(O)R^(b), —NHC(═O)NR^(b) ₂, —S(═O)₂—,—S(═O)₂OH, —S(═O)₂R^(b), —OS(═O)₂OR^(b), —S(═O)₂NR^(b) ₂, —S(═O)R^(b),—OP(═O)(OR^(b))₂, —P(═O)(ORN, —P(═O)(O)₂, —P(═O)(OH)₂, —P(O)(OR^(b))(O),—C(═O)R^(b), —C(═O)X, —C(S)R^(b), —C(O)OR^(b), —C(O)O⁻, —C(S)OR^(b),—C(O)SR^(b), —C(S)SR^(b), —C(O)NR^(b) ₂, —C(S)NR^(b) ₂,—C(═NR^(b))NR^(b) ₂, where each X is independently a halogen: F, Cl, Br,or I; and each R^(b) is independently H, alkyl, aryl, arylalkyl, aheterocycle, or a protecting group or prodrug moiety; R² is OR^(a); R³is halogen or N₃; each R^(a) is independently H, aryl, arylalkyl, or(C₁-C₈)alkyl; each of R⁴ and R⁵ is independently H, ═O, OR^(a),N(R^(a))₂, N₃, CN, S(O)_(n)R^(a), halogen, or (C₁-C₈)haloalkyl; each nis 0, 1 or 2; R⁶ is H, aryl, arylalkyl, or

wherein W¹ and W² are each, independently, Ole or a group of the FormulaIa:

wherein: each Y is independently a bond or O; M2 is 0, 1 or 2; eachR^(x) is H, halogen or OH.
 2. The compound of claim 1, represented byFormula II:

or a pharmaceutically acceptable salt, solvate, or ester thereof.
 3. Thecompound of claim 2, wherein R¹ is H.
 4. The compound of claim 3,wherein the compound is selected from the group consisting of

or a pharmaceutically acceptable salt, solvate, or ester thereof.
 5. Thecompound of claim 1, wherein R¹ is H, CH₂OH, CH₂F, CHF₂, CH═CH₂, C≡CH,CN, CH₂CH═CH₂, N₃, CH₃, or CH₂CH₃.
 6. The compound of claim 5, whereinR¹ is H.
 7. The compound of claim 1, wherein R² is OH or O-benzyl. 8.The compound of claim 7, wherein R² is OH.
 9. The compound of claim 1,wherein R³ is F or N₃.
 10. The compound of claim 9, wherein R³ is F. 11.The compound of claim 1, wherein R⁴ is NH₂ and R⁵ is H, F, Cl, Br, N₃,CN, CF₃, NH₂, SMe, or SO₂Me.
 12. The compound of claim 1, wherein R⁵ isNH₂ and R⁴ is ═O, OH, OMe, Cl, Br, I, NH₂, NHMe, NHcPr or SMe.
 13. Thecompound of claim 1, wherein each of R⁴ and R⁵ is independently selectedfrom the group consisting of H, NH₂, ═O, NHMe, NHcPr, OH, OMe, Cl, Br,I, SMe, F, N₃, CN, CF₃, and SO₂Me.
 14. The compound of claim 13, whereinR⁵ is H or NH₂.
 15. The compound of claim 13, wherein R⁴ is ═O or NH₂.16. The compound of claim 1, wherein R⁶ is H, benzyl, or

wherein W² is OH and W¹ is a group of the Formula Ia:

wherein: Y is O; M2 is 2; and each R^(x) is H.
 17. The compound of claim16, wherein R⁶ is H.
 18. The compound of claim 1, wherein R⁷ is H or OH.19. The compound of claim 18, wherein R⁷ is H.
 20. The compound of claim1, wherein R¹ is H, R² is OH and R³ is F.
 21. The compound of claim 20,wherein R⁴ and R⁵ are NH₂, H or ═O, and R⁶ and R⁷ are hydrogen.
 22. Thecompound of claim 1, wherein R¹ is H, R² is O-benzyl or OH, R³ is F, R⁴is SMe, NH₂ or ═O, R⁵ is SMe, SO₂Me, H or NH₂, R⁶ is benzyl

or wherein W² is OH and W¹ is a group of the Formula Ia:

wherein: Y is O; M2 is 2; and each R^(x) is H, and R⁷ is H or OH. 23.The compound of claim 1, wherein the compound is

or a pharmaceutically acceptable salt, solvate, or ester thereof. 24.The compound of claim 1, wherein the compound is

or a pharmaceutically acceptable salt, solvate, or ester thereof.
 25. Apharmaceutical composition comprising: a therapeutically effectiveamount of a compound of claim 1, and a pharmaceutically acceptablecarrier or excipient.
 26. The pharmaceutical composition of claim 25further comprising at least one additional therapeutic agent.
 27. Thepharmaceutical composition of claim 26, wherein the at least oneadditional therapeutic agent is selected from the group consisting of acorticosteroid, an anti-inflammatory signal transduction modulator, aβ2-adrenoreceptor agonist bronchodilator, an anticholinergic, amucolytic agent, hypertonic saline, an agent that inhibits migration ofpro-inflammatory cells to the site of infection, and mixtures thereof.28. The pharmaceutical composition of claim 27, wherein the at least oneadditional therapeutic agent is a viral haemagglutinin inhibitor, aviral neuramidase inhibitor, a M2 ion channel inhibitor, aOrthomyxoviridae RNA-dependent RNA polymerase inhibitor or a sialidase.29. The pharmaceutical composition of claim 27, wherein the at least oneadditional therapeutic agent is an interferon, ribavirin, oseltamivir,zanamivir, laninamivir, peramivir, amantadine, rimantadine, CS-8958,favipiravir, AVI-7100, alpha-1 protease inhibitor or DAS181.
 30. Amethod for treating an Orthomyxoviridae infection in a mammal in needthereof comprising administering a therapeutically effective amount of acompound of Formula I:

or a pharmaceutically acceptable salt, solvate, or ester thereof;wherein: each of R¹ and R⁷ is independently H, halogen, OR^(a),(C₁-C₈)haloalkyl, CN, N₃, (C₁-C₈)alkyl, (C₁-C₈)substituted alkyl,(C₂-C₈)alkenyl, (C₂-C₈)substituted alkenyl, (C₂-C₈)alkynyl or(C₂-C₈)substituted alkynyl, wherein the substituent is selected from thegroup consisting of —X, —R^(b), —OH, ═O, —OR^(b), —SR^(b), —S⁻, —NR^(b)₂, —N⁺R^(b) ₃, ═NR^(b), —CX₃, —CN, —OCN, —SCN, —N═C═O, —NCS, —NO, —NO₂,═N₂, —N₃, —NHC(═O)R^(b), —OC(O)R^(b), —NHC(═O)NR^(b) ₂, —S(˜0)₂—,—S(═O)₂OH, —S(═O)₂R^(b), —OS(═O)₂OR^(b), —S(═O)₂NR^(b) ₂, —S(═O)R^(b),—OP(═O)(ORN, —P(═O)(OR^(b))₂, —P(═O)(O⁻)₂, —P(═O)(OH)₂,—P(O)(OR^(b))(O), —C(═O)R^(b), —C(═O)X, —C(S)R^(b), —C(O)OR^(b),—C(O)O⁻, —C(S)OR^(b), —C(O)SR^(b), —C(S)SR^(b), —C(O)NR^(b) ₂,—C(S)NR^(b) ₂, —C(═NR^(b))NR^(b) ₂, where each X is independently ahalogen: F, Cl, Br, or I; and each R^(b) is independently H, alkyl,aryl, arylalkyl, a heterocycle, or a protecting group or prodrug moiety;R² is OR^(a); R³ is halogen or N₃; each R^(a) is independently H,arylalkyl, aryl, or (C₁-C₈)alkyl; each of R⁴ and R⁵ is independently H,═O, OR^(a), N(R^(a))₂, N₃, CN, S(O)_(n)R^(a), halogen, or(C₁-C₈)haloalkyl; each n is 0, 1 or 2; each R⁶ is H, aryl, arylalkyl, or

wherein W¹ and W² are each, independently, OR^(a) or a group of theFormula Ia:

wherein: each Y is independently a bond or O; M2 is 0, 1 or 2; eachR^(x) is H, halogen or OH.
 31. The method of claim 30, wherein thecompound of Formula I is represented by Formula II:

or a pharmaceutically acceptable salt, solvate, or ester thereof. 32.The method of claim 31, wherein R¹ is H.
 33. The method of claim 30,wherein R¹ is H, CH₂OH, CH₂F, CHF₂, CH═CH₂, C≡CH, CN, CH₂CH═CH₂, N₃, CH₃or CH₂CH₃.
 34. The method of claim 33, wherein R¹ is H.
 35. The methodof claim 30, wherein R² is OH or O-benzyl.
 36. The method of claim 35,wherein R² is OH.
 37. The method of claim 30, wherein R³ is F or N₃. 38.The method of claim 37, wherein R³ is F.
 39. The method of claim 30,wherein R⁴ is NH₂ and R⁵ is H, F, Cl, Br, N₃, CN, CF₃, NH₂, SMe, orSO₂Me.
 40. The method of claim 30, wherein R⁵ is NH₂ and R⁴ is ═O, OH,OMe, Cl, Br, I, NH₂, NHMe, NHcPr or SMe.
 41. The method of claim 30,wherein R⁴ and R⁵ are independently selected from the group consistingof H, NH₂, ═O, NHMe, NHcPr, OH, OMe, Cl, Br, I, SMe, F, N₃, CN, CF₃, andSO₂Me.
 42. The method of claim 41, wherein R⁵ is H or NH₂.
 43. Themethod of claim 41, wherein R⁴ is ═O or NH₂.
 44. The method of claim 30,wherein R⁶ is H, benzyl, or

wherein W² is OH and W¹ is a group of the Formula Ia:

wherein: each Y is O; M2 is 2; and each R^(x) is H.
 45. The method ofclaim 44, wherein R⁶ is H.
 46. The method of claim 30, wherein R⁷ is Hor OH.
 47. The method of claim 46, wherein R⁷ is H.
 48. The method ofclaim 30, wherein R¹ is H, R² is OH and R³ is F.
 49. The method of claim48, wherein R⁴ and R⁵ are NH₂, H or ═O, and R⁶ and R⁷ are hydrogen. 50.The method of claim 30, wherein R¹ is H, R² is O-benzyl or OH, R³ is F,R⁴ is SMe, NH₂ or ═O, R⁵ is SMe, SO₂Me, H or NH₂, R⁶ is benzyl or

wherein W² is OH and W¹ is a group of the Formula Ia:

wherein: Y is O; M2 is 2; and each R^(x) is H, and R⁷ is H or OH. 51.The method of claim 30, wherein the compound is

or a pharmaceutically acceptable salt, solvate, or ester thereof. 52.The method of claim 51, wherein the compound is

or a pharmaceutically acceptable salt, solvate, or ester thereof. 53.The method of claim 30 wherein the compound is

or a pharmaceutically acceptable salt, solvate, or ester thereof. 54.The method of claim 30 further comprising administering apharmaceutically acceptable carrier or excipient.
 55. The method ofclaim 30 further comprising administering a therapeutically effectiveamount of at least one additional therapeutic agent or compositionthereof selected from the group consisting of a corticosteroid, ananti-inflammatory signal transduction modulator, a β2-adrenoreceptoragonist bronchodilator, an anticholinergic, a mucolytic agent,hypertonic saline, an agent that inhibits migration of pro-inflammatorycells to the site of infection, and mixtures thereof.
 56. The method ofclaim 55, wherein the at least one additional therapeutic agent is aviral haemagglutinin inhibitor, a viral neuramidase inhibitor, a M2 ionchannel inhibitor, a Orthomyxoviridae RNA-dependent RNA polymeraseinhibitor or a sialidase.
 57. The method of claim 55, wherein the atleast one additional therapeutic agent is an interferon, ribavirin,oseltamivir, zanamivir, laninamivir, peramivir, amantadine, rimantadine,CS-8958, favipiravir, AVI-7100, alpha-1 protease inhibitor or DAS181.58. The method of claim 55, wherein the compound of Formula I, FormulaII and/or at least one therapeutic agent or mixtures thereof isadministered by inhalation.
 59. The method of claim 58, wherein thecompound of Formula I, Formula II and/or at least one therapeutic agentor mixtures thereof is administered by nebulization.
 60. The method ofclaim 30 wherein the Orthomyxoviridae infection is caused by anInfluenza A virus.
 61. The method of claim 30, wherein theOrthomyxoviridae infection is caused by an Influenza B virus.
 62. Themethod of claim 30, wherein the Orthomyxoviridae infection is caused byan Influenza C virus.
 63. The method of claim 30, wherein theOrthomyxoviridae infection is being treated by administering atherapeutically effective amount of a pharmaceutical compositioncomprising an effective amount of a Formula I compound, or apharmaceutically acceptable salt, solvate or ester thereof, incombination with a pharmaceutically acceptable diluent or carrier. 64.The method of claim 30, wherein a Orthomyxoviridae RNA-dependent RNApolymerase is inhibited.